U.S. patent application number 12/503135 was filed with the patent office on 2010-01-21 for novel derivatives.
Invention is credited to Jag Paul Heer, David Norton, Simon E. Ward.
Application Number | 20100016330 12/503135 |
Document ID | / |
Family ID | 39737241 |
Filed Date | 2010-01-21 |
United States Patent
Application |
20100016330 |
Kind Code |
A1 |
Heer; Jag Paul ; et
al. |
January 21, 2010 |
Novel Derivatives
Abstract
The present invention relates to novel piperazine derivatives;
to processes for their preparation; to pharmaceutical compositions
containing the derivatives; and to the use of the derivatives in
therapy to treat diseases for which blocking the Ca.sub.v2.2
calcium channels is beneficial.
Inventors: |
Heer; Jag Paul; (Harlow,
GB) ; Norton; David; (Harlow, GB) ; Ward;
Simon E.; (Harlow, GB) |
Correspondence
Address: |
GLAXOSMITHKLINE;CORPORATE INTELLECTUAL PROPERTY, MAI B482
FIVE MOORE DR., PO BOX 13398
RESEARCH TRIANGLE PARK
NC
27709-3398
US
|
Family ID: |
39737241 |
Appl. No.: |
12/503135 |
Filed: |
July 15, 2009 |
Current U.S.
Class: |
514/252.16 ;
544/281 |
Current CPC
Class: |
A61P 25/00 20180101;
A61P 25/04 20180101; A61P 43/00 20180101; C07D 487/04 20130101;
A61P 29/00 20180101; A61P 25/06 20180101; A61P 25/28 20180101 |
Class at
Publication: |
514/252.16 ;
544/281 |
International
Class: |
A61K 31/496 20060101
A61K031/496; C07D 487/04 20060101 C07D487/04; A61P 29/00 20060101
A61P029/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 17, 2008 |
GB |
0813144.3 |
Claims
1. A compound of formula (I), or a salt thereof, ##STR00065##
R.sup.1 is C.sub.1-4 alkyl, halogen or cyano; m and n are selected
from 0, 1 and 2; R.sup.2 is C.sub.1-4 alkyl; R.sup.3 is hydrogen,
cyano, C.sub.1-4 haloalkyl, C.sub.1-4 haloalkoxy or halogen;
R.sup.4 is hydrogen or C.sub.1-4 alkyl; R.sup.5 is hydrogen, cyano,
C.sub.1-4 haloalkyl, C.sub.1-4 haloalkoxy or halogen; R.sup.6 is
hydrogen, cyano, C.sub.1-4 haloalkyl, C.sub.1-4 haloalkoxy or
halogen; such that at least 1 of R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 is a group other than hydrogen; R.sup.7 is hydrogen or
C.sub.1-4 alkyl; such that when R.sup.7 is C.sub.1-4 alkyl, n is
0.
2. The compound or salt according to claim 1, wherein n is 0 or
1.
3. The compound or salt according to claim 2, wherein n is 0.
4. The compound or salt according to claim 1, wherein R.sup.1 is
selected from methyl, fluoro, chloro and cyano.
5. The compound or salt according to claim 4, wherein R.sup.1 is
selected from methyl and fluoro.
6. The compound or salt according to claim 1, wherein R.sup.7 is
selected from hydrogen and methyl.
7. The compound or salt according to claim 6, wherein R.sup.7 is
hydrogen.
8. The compound or salt according to claim 1, wherein R.sup.2 is
methyl.
9. The compound or salt according to claim 8, wherein R.sup.2 is
methyl and m is 1.
10. The compound or salt according to claim 9, wherein the compound
is a compound of formula (Ib) ##STR00066## , or a salt thereof.
11. The compound or salt according to claim 1, wherein R.sup.3 is
selected from trifluoromethyl, cyano, trifluoromethoxy and
hydrogen.
12. The compound or salt according to claim 11, wherein R.sup.3 is
selected from trifluoromethyl and trifluoromethoxy.
13. The compound or salt according to claim 12, wherein R.sup.3 is
trifluoromethyl.
14. The compound or salt according to claim 1, wherein R.sup.4 is
hydrogen or methyl.
15. The compound or salt according to claim 14, wherein R.sup.4 is
hydrogen.
16. The compound or salt according to claim 1, wherein R.sup.5 and
R.sup.6 are independently selected from hydrogen and C.sub.1-4
haloalkyl.
17. The compound or salt according to claim 16, wherein R.sup.5 and
R.sup.6 are independently selected from hydrogen and
trifluoromethyl.
18. The compound or salt according to claim 17, wherein R.sup.5 and
R.sup.6 are hydrogen.
19. The compound or salt according to claim 1, of formula
##STR00067## wherein R.sup.1 represents C.sub.1-4 alkyl; n
represents an integer from 0 or 1; m represents an integer from 0
to 1; R.sup.2 represents C.sub.1-4 alkyl; R.sup.3 represents cyano,
trifluoromethyl or trifluoromethoxy; R.sup.4 represents hydrogen or
methyl; such that when R.sup.3 represents cyano, R.sup.4 represents
a group other than hydrogen; or a salt thereof.
20. The compound, or a pharmaceutically acceptable salt thereof,
according to claim 1, which is
3-[((2S)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)c-
arbonyl]pyrazolo[1,5-a]pyrimidine of formula ##STR00068## , or a
salt thereof.
21. The compound or salt according to claim 1, wherein the salt is
a pharmaceutically acceptable salt.
22. A pharmaceutical composition comprising (a) a compound or salt
according to claim 21, and (b) a pharmaceutically acceptable
excipient.
23. A compound according to claim 21 for use in therapy.
24. A compound according to claim 23 for use in the treatment of
pain.
25. A method for the treatment of pain in a human in need thereof
comprising administering to said human a therapeutically effective
amount of a compound according to claim 21.
Description
[0001] This application claims priority to GB Application No.
0813144.3 filed Jul. 17, 2008 in the United Kingdom, the disclosure
of which is incorporate herein by reference in its entirety.
[0002] The present invention relates to novel piperazine
derivatives; to processes for their preparation; to pharmaceutical
compositions containing the derivatives; and to the use of the
derivatives in therapy to treat diseases for which blocking the
Ca.sub.v2.2 calcium channels is beneficial.
[0003] Pre-synaptic Ca.sub.v2.2 (N-type) voltage-gated calcium
channels in the dorsal horn of the spinal cord modulate the release
of key pro-nociceptive neurotransmitters such as glutamate,
substance P (SP) and calcitonin-gene-related peptide (CGRP),
indicating the potential therapeutic use of Ca.sub.v2.2 calcium
channel blockers as analgesics.
[0004] Peptidic .omega.-conotoxins, isolated from the venom of cone
snails, have been shown to be selective for Ca.sub.v2.2 calcium
channels and can block SP release in the spinal cord (Smith et al.
(2002) Pain, 96: 119-127). Moreover, they have been shown to be
antinociceptive in animal models of chronic pain following
intrathecal administration (Bowersox et al. (1996) Journal of
Pharmacology and Experimental Therapeutics, 279: 1243-1249; Smith
et al. (2002) supra), and have been shown to be effective
analgesics in clinical use, particularly in the treatment of
neuropathic pain (Brose et al. (1997) Clinical Journal of Pain, 13:
256-259).
[0005] In addition, Ca.sub.v2.2 calcium channels have been shown to
be important for normal neuronal function (Winquist et al. (2005)
Biochemical Pharmacology, 70: 489-499). Therefore, the aim is to
identify novel molecules that preferentially block Ca.sub.v2.2
under conditions of increased neuronal excitability, so-called
use-dependent blockers, as is the case in chronic pain
syndromes.
[0006] WO 2007/111921 (Amgen Inc) describes a series of
diazaheterocyclic amide derivatives which are claimed to be useful
in the treatment of diabetes, obesity and related conditions and
disorders. WO 2008/024284 (Merck & Co) describes a series of
sulfonylated piperazines as cannabinoid-1 (CB1) receptor modulators
which are claimed to be useful in the treatment for example of
psychosis, cognitive disorders and Alzheimer's disease.
[0007] The present invention provides compounds which are capable
of blocking these Ca.sub.v2.2 calcium channels.
[0008] In a first aspect, there is provided a compound of formula
(I), or a salt thereof, wherein:
##STR00001##
[0009] R.sup.1 is C.sub.1-4 alkyl, halogen or cyano;
[0010] m and n are selected from 0, 1 and 2;
[0011] R.sup.2 is C.sub.1-4 alkyl;
[0012] R.sup.3 is hydrogen, cyano, C.sub.1-4 haloalkyl, C.sub.1-4
haloalkoxy or halogen;
[0013] R.sup.4 is hydrogen or C.sub.1-4 alkyl;
[0014] R.sup.5 is hydrogen, cyano, C.sub.1-4 haloalkyl, C.sub.1-4
haloalkoxy or halogen;
[0015] R.sup.6 is hydrogen, cyano, C.sub.1-4 haloalkyl, C.sub.1-4
haloalkoxy or halogen;
[0016] such that at least 1 of R.sup.3, R.sup.4, R.sup.5 and
R.sup.6 is a group other than hydrogen;
[0017] R.sup.7 is hydrogen or C.sub.1-4 alkyl; such that when
R.sup.7 is C.sub.1-4 alkyl, n is 0.
[0018] It is understood that in formula (I), when present, R.sup.1
may be attached to any one of the three possible carbon atoms in
the 6 membered ring.
[0019] As used herein, the term "alkyl" (when used as a group or as
part of a group) refers to a straight or branched hydrocarbon chain
containing the specified number of carbon atoms. For example,
C.sub.1-4 alkyl means a straight or branched hydrocarbon chain
containing at least 1 and at most 4 carbon atoms. Compounds of
alkyl include, but are not limited to; methyl (Me), ethyl (Et),
n-propyl, i-propyl, t-butyl, n-butyl and i-butyl.
[0020] As used herein, the term "alkoxy" (when used as a group or
as part of a group) refers to an --O-alkyl group wherein alkyl is
as defined hereinbefore.
[0021] The term `halogen` is used herein to describe, unless
otherwise stated, a group selected from fluoro (fluorine), chloro
(chlorine), bromo (bromine) or iodo (iodine).
[0022] The term C.sub.1-4 haloalkyl as used herein refers to a
C.sub.1-4 alkyl group as defined herein substituted with one or
more halogen groups, e.g. CF.sub.3, CF.sub.2H or
CF.sub.3CH.sub.2.
[0023] The term C.sub.1-4 haloalkoxy as used herein refers to an
C.sub.1-4 alkoxy group as defined herein substituted with one or
more halogen groups, e.g. --O--CF.sub.3.
[0024] In one embodiment of the first aspect, n is 0 or 1. In a
particular embodiment of the first aspect, n is 0.
[0025] In one embodiment of the first aspect, when present, R.sup.1
is selected from methyl, fluoro, chloro and cyano. In a particular
embodiment of the first aspect, R.sup.1 is selected from methyl and
fluoro. In a more particular embodiment of the first aspect,
R.sup.1 is selected from methyl.
[0026] In one embodiment of the first aspect, R.sup.7 is selected
from hydrogen and methyl. In a particular embodiment of the first
aspect, R.sup.7 is hydrogen.
[0027] In one embodiment of the first aspect, when present, R.sup.2
is methyl. In a particular embodiment of the first aspect, R.sup.2
is methyl and m is 1. In a more particular embodiment of the first
aspect, the compound of formula (I) is a compound of formula
(Ia)
##STR00002##
[0028] In an even more particular embodiment of the first or second
aspect, the compound of formula (I) is a compound of formula
(Ib)
##STR00003##
[0029] In one embodiment of the first aspect, R.sup.3 is selected
from trifluoromethyl, cyano, trifluoromethoxy and hydrogen. In a
particular embodiment of the first aspect, R.sup.3 is selected from
trifluoromethyl and trifluoromethoxy. In a more particular
embodiment of the first aspect, R.sup.3 is trifluoromethyl.
[0030] In a particular embodiment of the first aspect, R.sup.4 is
hydrogen or methyl. In a more particular embodiment of the first
aspect, R.sup.4 is hydrogen.
[0031] In a particular embodiment of the first aspect, R.sup.5 and
R.sup.6 are independently selected from hydrogen and C.sub.1-4
haloalkyl. In a particular embodiment of the first aspect, R.sup.5
and R.sup.6 are independently selected from hydrogen and
trifluoromethyl. In a more particular embodiment of the first
aspect, R.sup.5 and R.sup.6 are hydrogen.
[0032] In one embodiment of the first aspect, n is 0 or 1; when
present, R.sup.1 is selected from methyl, fluoro, chloro and cyano;
R.sup.7 is selected from hydrogen and methyl; when present, R.sup.2
is methyl; R.sup.3 is selected from trifluoromethyl, cyano,
trifluoromethoxy and hydrogen; R.sup.4 is hydrogen or methyl; and
R.sup.5 and R.sup.6 are independently selected from hydrogen and
C.sub.1-4 haloalkyl.
[0033] In one embodiment of the first aspect, n is 0; R.sup.7 is
hydrogen; R.sup.2 is methyl and m is 1; R.sup.3 is selected from
trifluoromethyl and trifluoromethoxy; R.sup.4 is hydrogen; and
R.sup.5 and R.sup.6 are independently selected from hydrogen and
trifluoromethyl.
[0034] In one embodiment of the first aspect, n is 0; R.sup.7 is
hydrogen; R.sup.2 is 2-methyl in relation to the piperazine
carbonyl bond (as in formula (Ia)) and m is 1; R.sup.3 is selected
from trifluoromethyl and trifluoromethoxy; R.sup.4 is hydrogen; and
R.sup.5 and R.sup.6 are hydrogen.
[0035] In a second aspect, the compound of formula (I) is a
compound of formula (Ic), or a salt thereof, wherein:
##STR00004##
[0036] R.sup.1 represents C.sub.1-4 alkyl;
[0037] n represents an integer from 0 or 1;
[0038] m represents an integer from 0 to 1;
[0039] R.sup.2 represents C.sub.1-4 alkyl;
[0040] R.sup.3 represents cyano, trifluoromethyl or
trifluoromethoxy;
[0041] R.sup.4 represents hydrogen or methyl;
[0042] such that when R.sup.3 represents cyano, R.sup.4 represents
a group other than hydrogen.
[0043] In one embodiment of the second aspect, n represents 0 or 1.
In a further embodiment, n represents 0. When present, in one
embodiment of the second aspect, R.sup.1 represents C.sub.1-4 alkyl
(e.g. methyl).
[0044] In one embodiment of the second aspect, m represents 0 or 1.
In a further embodiment of the second aspect, m represents 1.
[0045] When present, in one embodiment of the second aspect,
R.sup.2 represents C.sub.1-3 alkyl. In a further embodiment of the
second aspect, R.sup.2 represents methyl or ethyl. In a yet further
embodiment of the second aspect, R.sup.2 represents methyl.
[0046] In one embodiment of the second aspect, R.sup.3 represents
trifluoromethyl.
[0047] In one embodiment of the first or second aspect, the
compound is selected from a compound of Examples 1 to 24, or a salt
thereof.
[0048] In one embodiment of the first or second aspect, a compound
is selected from:
[0049]
3-[(4-{[4-(Trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)carbonyl]-
pyrazolo[1,5-a]pyrimidine;
[0050]
3-[((2S)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperaz-
inyl)carbonyl]pyrazolo[1,5-a]pyrimidine;
[0051]
3-[((3R)-3-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperaz-
inyl)carbonyl]pyrazolo[1,5-a]pyrimidine;
[0052]
3-[((3S)-3-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperaz-
inyl)carbonyl]pyrazolo[1,5-a]pyrimidine;
[0053]
3-Methyl-4-{[(3S)-3-methyl-4-(pyrazolo[1,5-a]pyrimidin-3-ylcarbonyl-
)-1-piperazinyl]sulfonyl}benzonitrile;
[0054]
6-Methyl-3-[(4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)-
carbonyl]pyrazolo[1,5-a]pyrimidine; and
[0055]
3-[((2R)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperaz-
inyl)carbonyl]pyrazolo[1,5-a]pyrimidine; or a salt thereof;
[0056] or a salt thereof.
[0057] In one embodiment of the first and second aspect, the
compound is:
[0058]
3-[((2S)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperaz-
inyl)carbonyl]pyrazolo[1,5-a]pyrimidine; or
[0059]
3-[((2R)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperaz-
inyl)carbonyl]pyrazolo[1,5-a]pyrimidine;
[0060] or a salt thereof.
[0061] Certain compounds as defined in the first or second aspect
may in some circumstances form acid addition salts thereof. It will
be appreciated that for use in medicine compounds as defined in the
first or second aspect may be used as salts, in which case the
salts should be pharmaceutically acceptable. Pharmaceutically
acceptable salts include those described by Berge, Bighley and
Monkhouse, J. Pharm. Sci., 1977, 66, 1-19. The term
"pharmaceutically acceptable salts" includes salts prepared from
pharmaceutically acceptable acids, including inorganic and organic
acids. Such acids include acetic, benzenesulfonic, benzoic,
camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic,
glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic,
malic, mandelic, methanesulfonic, mucic, nitric, pamoic,
pantothenic, phosphoric, succinic, sulfuric, tartaric,
p-toluenesulfonic acid, and the like.
[0062] Compounds of pharmaceutically acceptable salts include those
formed from maleic, fumaric, benzoic, ascorbic, pamoic, succinic,
hydrochloric, sulfuric, bismethylenesalicylic, methanesulfonic,
ethanedisulfonic, propionic, tartaric, salicylic, citric, gluconic,
aspartic, stearic, palmitic, itaconic, glycolic, p-aminobenzoic,
glutamic, benzenesulfonic, cyclohexylsulfamic, phosphoric and
nitric acids.
[0063] It will be appreciated by those skilled in the art that
certain protected derivatives of the compounds as defined in the
first or second aspect, which may be made prior to a final
deprotection stage, may not possess pharmacological activity as
such, but may, in certain instances, be administered orally or
parenterally and thereafter metabolized in the body to form
compounds as defined in the first and second aspect which are
pharmacologically active. Such derivatives may therefore be
described as "prodrugs". All protected derivatives and prodrugs of
compounds defined in the first and second aspect are included
within the scope of the invention. Compounds of suitable pro-drugs
for the compounds of the present invention are described in Drugs
of Today, Volume 19, Number 9, 1983, pp 499-538 and in Topics in
Chemistry, Chapter 31, pp 306-316 and in "Design of Prodrugs" by H.
Bundgaard, Elsevier, 1985, Chapter 1 (the disclosures in which
documents are incorporated herein by reference). It will further be
appreciated by those skilled in the art, that certain moieties,
known to those skilled in the art as "pro-moieties", for example as
described by H. Bundgaard in "Design of Prodrugs" (the disclosure
in which document is incorporated herein by reference) may be
placed on appropriate functionalities when such functionalities are
present within the compounds as defined in the first and second
aspects. Therefore, in a further aspect, the invention provides a
prodrug of a compound as defined in the first and second
aspect.
[0064] It will be appreciated that certain compounds as defined in
the first or second aspect, or their salts, may exist as solvates,
such as hydrates. Where solvates exist, this invention includes
within its scope stoichiometric and non-stoichiometric
solvates.
[0065] It will be appreciated that certain compounds as defined in
the first or second aspect, or their salts, may exist in more than
one polymorphic form. The invention extends to all such forms
whether in a pure polymorphic form or when admixed with any other
material, such as another polymorphic form.
[0066] Certain compounds as defined in the first or second aspect
are capable of existing in stereoisomeric forms (e.g. diastereomers
and enantiomers) and the invention extends to each of these
stereoisomeric forms and to mixtures thereof including racemates.
The different stereoisomeric forms may be separated one from the
other by the usual methods, or any given isomer may be obtained by
stereospecific or asymmetric synthesis. The invention also extends
to any tautomeric forms and mixtures thereof.
[0067] The subject invention also includes isotopically-labelled
compounds, which are identical to the compounds as defined in the
first or second aspect, but for the fact that one or more atoms are
replaced by an atom having an atomic mass or mass number different
from the atomic mass or mass number most commonly found in nature.
Compounds of isotopes that can be incorporated into compounds of
the invention include isotopes of hydrogen, carbon, nitrogen,
fluorine, such as .sup.3H, .sup.11C, .sup.14C and .sup.18F.
[0068] Compounds as defined in the first or second aspect and salts
of said compounds that contain the aforementioned isotopes and/or
other isotopes of other atoms are within the scope of the present
invention. Isotopically-labelled compounds of the present
invention, for example those into which radioactive isotopes such
as .sup.3H, .sup.14C are incorporated, are useful in drug and/or
substrate tissue distribution assays. Tritiated, i.e., .sup.3H, and
carbon-14, i.e., .sup.14C, isotopes are particularly preferred for
their ease of preparation and detectability. .sup.11C and .sup.18F
isotopes are particularly useful in PET (positron emission
tomography). PET is useful in brain imaging. Further, substitution
with heavier isotopes such as deuterium, i.e., .sup.2H, can afford
certain therapeutic advantages resulting from greater metabolic
stability, for example increased in vivo half-life or reduced
dosage requirements and, hence, may be preferred in some
circumstances. Isotopically labelled compounds as defined in the
first or second aspect and following of this invention can
generally be prepared by carrying out the procedures disclosed in
the Schemes and/or in the Compounds below, by substituting a
readily available isotopically labelled reagent for a
non-isotopically labelled reagent. In one embodiment, compounds as
defined in the first or second aspect, or salts thereof, are not
isotopically labelled.
[0069] A compound of formula (I) as defined in the first or second
aspect may be prepared as set forth in the following Schemes and in
the experimental write-ups. The following processes form another
aspect of the present invention.
[0070] Throughout the specification, general formulae are
designated by Roman numerals (I), (II), (III), (IV), etc. Subsets
of these general formulae are defined as (Ia), (Ib), (Ic), etc. . .
. (IVa), (IVb), (IVc), etc.
[0071] The present invention also provides a process for the
preparation of a compound as defined in the first or second aspect,
or a salt thereof, which process comprises:
[0072] (a) reacting a compound of formula (II)
##STR00005##
[0073] or a derivative thereof, with a compound of formula
(III)
##STR00006##
[0074] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, m and n are as defined above and L.sup.1
represents a suitable leaving group such as a halogen atom (e.g.
chlorine or bromine) or hydroxyl group activated by commercially
available amide coupling reagents (for example HOBT, HBTU and
HATU);
[0075] (b) reacting a compound of formula (IV)
##STR00007##
[0076] with a compound of formula (V)
##STR00008##
[0077] wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, m and n are as defined above and L.sup.2
represents a suitable leaving group such as a halogen atom (e.g.
chlorine or bromine);
[0078] (c) interconversion to other compounds as defined in the
first or second aspect.
[0079] Process (a) typically comprises reaction of a compound of
formula (II) with a compound of formula (III) in a suitable solvent
such as acetonitrile, tetrahydrofuran, N,N-dimethylformamide or
dichloromethane, in the presence of a suitable base, (for example,
triethylamine, di-isopropylethylamine or PS-DIPEA) at 0.degree. C.
to ambient temperature (for example, room temperature).
[0080] Process (b) typically comprises reaction of a compound of
formula (IV) and (V) in the presence of a suitable solvent (such as
dichloromethane or acetonitrile) in the presence of a suitable
base, (for example triethylamine, di-isopropylethylamine or
PS-DIPEA) at 0.degree. C. to ambient temperature (for example, room
temperature). Alternatively, process (b) may typically comprise
reaction of the Descriptions in the presence of a suitable base as
a solvent (for example pyridine).
[0081] Process (c) may be performed using conventional
interconversion procedures such as epimerization, oxidation,
reduction, alkylation, nucleophilic or electrophilic aromatic
substitution.
[0082] For example, a compound as defined in the first or second
aspect, where R.sup.1 is bromo, may be converted to a compound as
defined in the first or second aspect where R.sup.1 is a nitrile
(cyano) group. This step typically comprises reacting the bromo
compound with zinc cyanide in the presence of a palladium catalyst
(for example tris(dibenzildeneacetone)dipalladium(0)) and ligand
(for example 1,1'-bis(diphenylphosphino)ferrocene) in a suitable
solvent (such as N,N-dimethylformamide) at elevated temperatures
(such as 95.degree. C.).
[0083] Another example of an interconversion is from a compound of
formula (VII) where R.sup.3 is bromo to a compound of formula (VII)
where R.sup.3 is a nitrile (cyano) group. This step typically
comprises reacting the bromo compound with zinc cyanide in the
presence of a palladium catalyst (for example
tris(dibenzildeneacetone)dipalladium(0)) and ligand (for example
1,1'-bis(diphenylphosphino)ferrocene) in a suitable solvent (such
as N,N-dimethylformamide) at elevated temperatures (such as
120.degree. C.).
[0084] Compounds of formula (II) may be prepared in accordance with
the following Scheme:
##STR00009##
[0085] wherein R.sup.2, R.sup.3, R.sup.4, m and L.sup.2 are as
defined above and P.sup.1 represents a suitable protecting group
such as t-butoxycarbonyl.
[0086] Step (i) typically comprises reacting a compound of formula
(V) and (VI) in a suitable solvent, such as dichloromethane or
acetonitrile in the presence of a base, (for example triethylamine,
di-isopropylethylamine or PS-DIPEA) at 0.degree. C. to ambient
temperature (for example room temperature). Alternatively, step (i)
may typically be carried out using a suitable base as a solvent
(for example pyridine).
[0087] Step (ii) typically comprises a deprotection reaction. For
example, when P.sup.1 represents t-butoxycarbonyl, step (ii) will
typically comprise treatment with an acid, for example hydrochloric
acid or trifluoroacetic acid, in a solvent (such as 1,4-dioxane,
dichloromethane or a mixture of methanol and 1,4-dioxane).
[0088] Compounds of formula (IV) may be prepared in accordance with
the following Scheme:
##STR00010##
[0089] wherein R.sup.2, m, R.sup.1, n and P.sup.1 are as defined
above.
[0090] Step (i) typically comprises reacting a compound of formula
(VI) with a compound of formula (III) in a suitable solvent (such
as acetonitrile, tetrahydrofuran, N,N-dimethylformamide or
dichloromethane) in the presence of a suitable base (for example,
triethylamine, di-isopropylethylamine or PS-DIPEA) at 0.degree. C.
to ambient temperature (for example room temperature).
[0091] Step (ii) typically comprises a deprotection reaction which
may be carried out in an analogous manner to Step (ii) above.
[0092] Compounds of formula (III), (V) and (VI) are either
commercially available, or may be prepared by known methods.
[0093] For example, compounds of formula (III) where R.sup.1 is
chloro or bromo may be prepared according to the following
Scheme:
##STR00011##
[0094] wherein R.sup.1 is chloro or bromo.
[0095] Step (i) typically comprises reacting a compound of formula
(IX) with sodium hydroxide in the presence of solvents such as
ethanol and water.
[0096] Step (ii) involves heating a compound of formula (X) with
bromo- or chloromalonaldehyde in a suitable solvent (such as a
mixture of ethanol and acetic acid) at an elevated temperature
(such as 70.degree. C.).
[0097] Synthesis of a lithium salt of a compound of formula (III)
containing fluorine was achieved by initial formation of compound
(XI) following the method of K. Funabiki, T. Ohtsuki, T. Ishihara
and H. Yamanaka, Chem. Lett., 1994, 1075-78. Formation of compound
(IIIb) was then achieved by hydrolysis of (XI) with lithium
hydroxide in the microwave using a mixture of water, methanol and
THF.
##STR00012##
[0098] Compounds with affinity for Ca.sub.v2.2 calcium channels may
be useful in the treatment or prophylaxis of pain, including acute
pain, chronic pain, chronic articular pain, musculoskeletal pain,
neuropathic pain, inflammatory pain, visceral pain, pain associated
with cancer, pain associated with migraine, tension headache and
cluster headaches, pain associated with functional bowel disorders,
lower back and neck pain, pain associated with sprains and strains,
sympathetically maintained pain; myositis, pain associated with
influenza or other viral infections such as the common cold, pain
associated with rheumatic fever, pain associated with myocardial
ischemia, post operative pain, cancer chemotherapy, headache,
toothache and dysmenorrhea.
[0099] `Chronic articular pain` conditions include rheumatoid
arthritis, osteoarthritis, rheumatoid spondylitis, gouty arthritis
and juvenile arthritis.
[0100] `Pain associated with functional bowel disorders` includes
non-ulcer dyspepsia, non-cardiac chest pain and irritable bowel
syndrome.
[0101] `Neuropathic pain` syndromes include: diabetic neuropathy,
sciatica, non-specific lower back pain, trigeminal neuralgia,
multiple sclerosis pain, fibromyalgia, HIV-related neuropathy,
post-herpetic neuralgia, trigeminal neuralgia, and pain resulting
from physical trauma, amputation, phantom limb syndrome, spinal
surgery, cancer, toxins or chronic inflammatory conditions. In
addition, neuropathic pain conditions include pain associated with
normally non-painful sensations such as "pins and needles"
(paraesthesias and dysesthesias), increased sensitivity to touch
(hyperesthesia), painful sensation following innocuous stimulation
(dynamic, static, thermal or cold allodynia), increased sensitivity
to noxious stimuli (thermal, cold, mechanical hyperalgesia),
continuing pain sensation after removal of the stimulation
(hyperpathia) or an absence of or deficit in selective sensory
pathways (hypoalgesia).
[0102] Other conditions which could potentially be treated by
compounds of the present invention include neurodegenerative
diseases and neurodegeneration, neurodegeneration following trauma,
tinnitus, dependence on a dependence-inducing agent such as opiods
(e.g. morphine), CNS depressants (e.g. ethanol), psychostimulants
(e.g. cocaine) and nicotine.
[0103] Neurodegenerative diseases include dementia, particularly
degenerative dementia (including senile dementia, dementia with
Lewy bodies, Alzheimer's disease, Pick's disease, Huntingdon's
chorea, Parkinson's disease and Creutzfeldt-Jakob disease, ALS,
motor neuron disease); vascular dementia (including multi-infarct
dementia); as well as dementia associated with intracranial space
occupying lesions; trauma; infections and related conditions
(including HIV infection, meningitis and shingles); metabolism;
toxins; anoxia and vitamin deficiency; and mild cognitive
impairment associated with aging, particularly Age Associated
Memory Impairment.
[0104] The compounds as defined in the first and second aspect may
also be useful for neuroprotection and in the treatment or
prophylaxis of neurodegeneration following trauma such as stroke,
cardiac arrest, pulmonary bypass, traumatic brain injury, spinal
cord injury or the like.
[0105] Another condition which could potentially be treated by
compounds as defined in the first and second aspect is spasticity
or muscular hypertonicity.
[0106] Hence, in a third aspect, compounds as defined in the first
or second aspect, or a pharmaceutically salt thereof, are for use
in therapy.
[0107] In an embodiment of the third aspect, the therapy is to the
treatment or prophylaxis of any of the disorders described herein,
in particular pain. In one particular embodiment, the therapy is to
the treatment of any of the disorders described herein, in
particular pain.
[0108] According to a further aspect, there is provided a use of a
compound as defined in the first or second aspect, or a
pharmaceutically acceptable salt thereof, in the manufacture of a
medicament for the treatment or prophylaxis of any of the disorders
herein, in particular pain. More particularly, there is provided a
use of a compound of formula (I) as defined in the first and second
aspect, or a pharmaceutically acceptable salt thereof, in the
manufacture of a medicament for the treatment of any of the
disorders herein.
[0109] According to another aspect, there is provided a method of
treatment or prophylaxis of any of the disorders herein, in
particular pain in humans, which method comprises the
administration to the human in need of such treatment or
prophylaxis, an effective amount of a compound as defined in the
first or second aspect, or a pharmaceutically acceptable salt
thereof.
[0110] In the context of the present invention, the term
"treatment" refers to symptomatic treatment and the term
"prophylaxis" is used to mean preventing symptoms in an already
afflicted subject or preventing recurrence of symptoms in an
afflicted subject and is not limited to complete prevention of an
affliction.
[0111] In order to use a compound of formula (I) as defined in the
first and second aspect or a pharmaceutically acceptable salt
thereof for the treatment or prophylaxis of humans and other
mammals it is normally formulated in accordance with standard
pharmaceutical practice as a pharmaceutical composition. Therefore
in another aspect of the invention there is provided a
pharmaceutical composition comprising a compound of formula (I) as
defined in the first and second aspect, or a pharmaceutically
acceptable salt thereof, adapted for use in human or veterinary
medicine.
[0112] In order to use the compounds as defined in the first and
second aspect in therapy, they will normally be formulated into a
pharmaceutical composition in accordance with standard
pharmaceutical practice. The present invention also provides a
pharmaceutical composition, which comprises a compound of formula
(I) as defined in the first and second aspect, or a
pharmaceutically acceptable salt thereof, and optionally a
pharmaceutically acceptable excipient.
[0113] When used in the treatment or prophylaxis of pain, the
compound of formula (I) as defined in the first and second aspect
or a pharmaceutically acceptable salt thereof may be used in
combination with other medicaments indicated to be useful in the
treatment or prophylaxis of pain of neuropathic origin including
neuralgias, neuritis and back pain, and inflammatory pain including
osteoarthritis, rheumatoid arthritis, acute inflammatory pain, back
pain and migraine. Such therapeutic agents include for example
COX-2 (cyclooxygenase-2) inhibitors, such as celecoxib, deracoxib,
rofecoxib, valdecoxib, parecoxib, COX-189 or
2-(4-ethoxy-phenyl)-3-(4-methanesulfonyl-phenyl)-pyrazolo[1,5-b]pyridazin-
e (WO99/012930); 5-lipoxygenase inhibitors; NSAIDs (non-steroidal
anti-inflammatory drugs) such as diclofenac, indomethacin,
nabumetone or ibuprofen; bisphosphonates, leukotriene receptor
antagonists; DMARDs (disease modifying anti-rheumatic drugs) such
as methotrexate; adenosine A1 receptor agonists; sodium channel
blockers, such as lamotrigine; NMDA (N-methyl-D-aspartate) receptor
modulators, such as glycine receptor antagonists or memantine;
ligands for the .alpha..sub.2.delta.-subunit of voltage gated
calcium channels, such as gabapentin, pregabalin and solzira;
tricyclic antidepressants such as amitriptyline; neurone
stabilizing antiepileptic drugs; cholinesterase inhibitors such as
galantamine; mono-aminergic uptake inhibitors such as venlafaxine;
opioid analgesics; local anaesthetics; 5HT.sub.1 agonists, such as
triptans, for example sumatriptan, naratriptan, zolmitriptan,
eletriptan, frovatriptan, almotriptan or rizatriptan; nicotinic
acetyl choline (nACh) receptor modulators; glutamate receptor
modulators, for example modulators of the NR2B subtype; EP.sub.4
receptor ligands; EP.sub.2 receptor ligands; EP.sub.3 receptor
ligands; EP.sub.4 agonists and EP.sub.2 agonists; EP.sub.4
antagonists; EP.sub.2 antagonists and EP.sub.3 antagonists;
cannabinoid receptor ligands; bradykinin receptor ligands;
vanilloid receptor or Transient Receptor Potential (TRP) ligands;
and purinergic receptor ligands, including antagonists at
P2X.sub.3, P2X.sub.2/3, P2X.sub.4, P2X.sub.7 or P2X.sub.4/7;
KCNQ/Kv7 channel openers, such as retigabine; Additional COX-2
inhibitors are disclosed in U.S. Pat. No. 5,474,995, U.S. Pat. No.
5,633,272; U.S. Pat. No. 5,466,823, U.S. Pat. No. 6,310,099 and
U.S. Pat. No. 6,291,523; and in WO 96/25405, WO 97/38986, WO
98/03484, WO 97/14691, WO99/12930, WO00/26216, WO00/52008,
WO00/38311, WO01/58881 and WO02/18374.
[0114] When used in the treatment or prophylaxis of Alzheimer's
disease, the compound of formula (I) as defined in the first and
second aspect or a pharmaceutically acceptable salt thereof may be
used in combination with other medicaments indicated to be useful
as either disease modifying or symptomatic treatments of
Alzheimer's disease.
[0115] Suitable Compounds of such other therapeutic agents may be
agents known to modify cholinergic transmission such as 5-HT.sub.1A
antagonists, (e.g. lecozotan), 5-HT6 antagonists, M1 muscarinic
agonists, M2 muscarinic antagonist, acetylcholinesterase inhibitors
(e.g tetrahydroaminoacridine, donepezil or rivastigmine), or
allosteric modulators, nicotinic receptor agonists or allosteric
modulators, symptomatic agents such as 5-HT6 receptor antagonists,
e.g. SB742457, H3 receptor antagonists e.g. GSK189254 and
GSK239512, 5-HT4 receptor agonist, PPAR agonists, also NMDA
receptor antagonists or modulators, also disease modifying agents
such as .sup..about..beta. or .gamma.-secretase inhibitors (e.g.
R-flurbiprofen), also AMPA positive modulators and Glycine
Transporter Reuptake inhibitors.
[0116] When a compound of formula (I) as defined in the first and
second aspect or a pharmaceutically acceptable salt thereof is used
in combination with another therapeutic agent, the compounds may be
administered either sequentially or simultaneously by any
convenient route.
[0117] The invention thus provides, in a further aspect, a
combination comprising a compound of formula (I) as defined in the
first and second aspect or a pharmaceutically acceptable salt
thereof together with a further therapeutic agent or agents.
[0118] A pharmaceutical composition of the invention, which may be
prepared by admixture, suitably at ambient temperature and
atmospheric pressure, is usually adapted for oral, parenteral or
rectal administration and, as such, may be in the form of tablets,
capsules, oral liquid preparations, powders, granules, lozenges,
reconstitutable powders, injectable or infusable solutions or
suspensions or suppositories. Orally administrable compositions are
generally preferred.
[0119] Tablets and capsules for oral administration may be in unit
dose form, and may contain conventional excipients, such as binding
agents, fillers, tabletting lubricants, disintegrants and
acceptable wetting agents. The tablets may be coated according to
methods well known in normal pharmaceutical practice.
[0120] Oral liquid preparations may be in the form of, for example,
aqueous or oily suspension, solutions, emulsions, syrups or
elixirs, or may be in the form of a dry product for reconstitution
with water or other suitable vehicle before use. Such liquid
preparations may contain conventional additives such as suspending
agents, emulsifying agents, non-aqueous vehicles (which may include
edible oils), preservatives, and, if desired, conventional
flavourings or colourants.
[0121] For parenteral administration, fluid unit dosage forms are
prepared utilizing a compound of the invention or pharmaceutically
acceptable salt thereof and a sterile vehicle. The compound,
depending on the vehicle and concentration used, can be either
suspended or dissolved in the vehicle. In preparing solutions, the
compound can be dissolved for injection and filter sterilized
before filling into a suitable vial or ampoule and sealing.
Advantageously, adjuvants such as a local anaesthetic,
preservatives and buffering agents are dissolved in the vehicle. To
enhance the stability, the composition can be frozen after filling
into the vial and the water removed under vacuum. Parenteral
suspensions are prepared in substantially the same manner, except
that the compound is suspended in the vehicle instead of being
dissolved, and sterilization cannot be accomplished by filtration.
The compound can be sterilized by exposure to ethylene oxide before
suspension in a sterile vehicle. Advantageously, a surfactant or
wetting agent is included in the composition to facilitate uniform
distribution of the compound.
[0122] The composition may contain from 0.1% to 99% by weight,
preferably from 10% to 60% by weight, of the active material,
depending on the method of administration. The dose of the compound
of formula (I) as defined in the first and second aspect or a
pharmaceutically acceptable salt thereof used in the treatment or
prophylaxis of the aforementioned disorders will vary in the usual
way with the seriousness of the disorders, the weight of the
sufferer, and other similar factors. However, as a general guide
suitable unit doses may be 0.05 to 1000 mg, more suitably 1.0 to
200 mg, and such unit doses may be administered more than once a
day, for example two or three a day. Such therapy may extend for a
number of weeks, months, years or even life.
[0123] A further aspect to the invention is a pharmaceutical
composition comprising 0.05 to 1000 mg of a compound of formula (I)
or a pharmaceutically acceptable salt thereof, and 0 to 3 g more
suitably 0 to 2 g of at least one pharmaceutically acceptable
carrier.
[0124] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0125] Abbreviations: [0126] Ar--argon [0127] API--atmospheric
pressure ionization [0128] aq.--aqueous [0129] eq.--equivalent
[0130] dba--dibenzylideneacetone [0131] DCM--dichloromethane [0132]
DIPEA--diisopropylethylamine [0133] DMF--N,N-dimethylformamide
[0134] DMSO--dimethylsulfoxide [0135]
DPPF--1,1'-Bis(diphenylphosphino)ferrocene [0136] EtOAc--ethyl
acetate [0137] ES--Electrospray [0138]
HATU--o-(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate [0139]
HBTU--O-benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate
[0140] HOBT--Hydroxybenzotriazole [0141] HPLC--high performance
liquid chromatography [0142] LCMS--Liquid Chromatography Mass
Spectrometry [0143] MS--mass spectrometry [0144] MeCN--acetonitrile
[0145] MDAP--mass directed automated preparative liquid
chromatography. [0146] MeOH--methanol [0147] NMR--nuclear magnetic
resonance [0148] PS-DIPEA--polymer supported diisopropylethylamine
[0149] rt--room temperature [0150] sat.--saturated [0151]
SCX--strong cation exchange chromatography [0152]
THF--tetrahydrofuran [0153] TFA--trifluoroacetic acid [0154]
Pd.sub.2(dba).sub.3--tris(dibenzylideneacetone)dipalladium(0)
[0155] Pd(PPh.sub.3).sub.4--tetrakis(triphenylphosphine)palladium
[0156] UPLC--Ultra Performance Liquid Chromatography [0157]
h--hour(s) [0158] min--minutes
EXAMPLES
[0159] The preparation of a number of supporting compounds as
defined in the first to fourth aspect are described below.
[0160] In the procedures that follow, after each starting material,
reference to a Description is typically provided. This is provided
merely for assistance to the skilled chemist. The starting material
may not necessarily have been prepared from the batch referred
to.
[0161] Description 1
1-{[4-(Trifluoromethyl)phenyl]sulfonyl}piperazine
##STR00013##
[0163] To a solution of 1,1-dimethylethyl 1-piperazinecarboxylate
(5.00 g, 26.8 mmol) in DCM (200 ml) was added DIPEA (9.85 ml, 56.4
mmol) and then 4-(trifluoromethyl)benzenesulfonyl chloride (7.22 g,
29.5 mmol). The reaction mixture was stirred for 1.5 hours at rt.
The reaction mixture was then reduced to dryness in vacuo, to yield
1,1-dimethylethyl
4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinecarboxylate.
[0164] m/z (API-ES) 294.72 [M+H-100].sup.+
[0165] To a solution of 1,1-dimethylethyl
4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinecarboxylate in
1,4-dioxane (100 ml) was added 4M HCl in 1,4-dioxane (50 ml, 200
mmol) and 3 drops of distilled water. The reaction mixture was
stirred overnight and then reduced to dryness in vacuo. The residue
was dissolved in DCM (200 ml) and washed with 2M aq. NaOH (50 ml),
twice. The organic layer was dried over dried magnesium sulphate,
the insolubles removed by filtration, and filtrate reduced to
dryness in vacuo to yield the title compound (6.60 g) as a pale
yellow solid.
[0166] m/z (API-ES) 294.74 [M+H].sup.+
[0167] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 2.89-2.98
(m, 4H), 2.99-3.09 (m, 4H), 3.71 (s, 1H), 7.77-7.85 (m, 2H),
7.85-7.92 (m, 2H).
[0168] Description 2
(3S)-3-Methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
##STR00014##
[0170] To a solution of
1,1-dimethylethyl(2S)-2-methyl-1-piperazinecarboxylate (5.00 g,
25.0 mmol) in DCM (200 ml) was added DIPEA (11.4 ml, 65.5 mmol) and
4-(trifluoromethyl)benzenesulfonyl chloride (5.68 g, 23.2 mmol).
The reaction mixture was stirred for 1 hour. DCM (200 ml) was added
to the reaction mixture which was transferred to a separating
funnel. The solution was washed with saturated aq. sodium
bicarbonate solution (50 ml, twice) and then with distilled water
(50 ml). The organic layer was dried over magnesium sulphate which
was removed by filtration and the filtrate was evaporated to
dryness in vacuo to give 8.9 g of white solid.
[0171] The solid was dissolved in 1,4-dioxane (30 ml) and 4M HCl in
1,4-dioxane (10 ml) and a few drops of water were added and the
reaction mixture was stirred for 1 hour. Further 4M HCl in
1,4-dioxane (20 ml) was added and the reaction stirred
overnight.
[0172] The reaction mixture was evaporated to dryness in vacuo and
the residue was dissolved in MeOH (100 ml) and loaded onto an SCX
column (Biotage). The column was washed with MeOH and the product
was eluted with 1M ammonia in MeOH. LCMS showed a large amount of
desired product present in the MeOH wash, so this was evaporated to
dryness on the rotary evaporator. The residue was dissolved in
EtOAc (100 ml) and extracted with 2M HCl (50 ml). The aqueous layer
was basified with 2M aq. NaOH solution until pH remained above 7
and extracted with EtOAc (100 ml). The organic layer was evaporated
to dryness on the rotary evaporator to yield the title compound
(4.34 g).
[0173] m/z (API-ES) 309 [M+H].sup.+
[0174] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 1.36 (d, J=6.6
Hz, 3H), 2.62-2.73 (m, 1H), 2.85-2.97 (m, 1H), 3.19-3.29 (m, 1H),
3.45-3.54 (m, 2H), 3.80-3.95 (m, 2H), 7.95 (d, J=8.3 Hz, 2H), 8.05
(d, J=8.3 Hz, 2H).
[0175] Description 3
1,1-Dimethylethyl(3R)-3-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-p-
iperazinecarboxylate
##STR00015##
[0177] To a solution of
1,1-dimethylethyl(3R)-3-methyl-1-piperazinecarboxylate (1.50 g,
7.49 mmol) in DCM (30 ml) was added DIPEA (1.96 ml, 11.2 mmol) and
then portionwise addition of 4-(trifluoromethyl)benzenesulfonyl
chloride (2.20 g, 8.99 mmol) at rt. The resultant mixture was
stirred under an atmosphere of argon for 2 hours before addition of
1M aq. HCl solution (75 ml) and DCM (75 ml). The layers were
separated and the aqueous layer was then re-extracted with DCM (75
ml), the organic layers were combined and washed with saturated
brine solution (100 ml). The organic layers were then separated,
dried (MgSO.sub.4) and concentrated to dryness giving the title
compound (3.39 g).
[0178] m/z (API-ES) 309 [M+H-100].sup.+
[0179] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.03 (d,
J=6.7 Hz, 3H) 1.43 (s, 9H) 2.65-3.22 (m, 3H) 3.54-4.27 (m, 4H) 7.78
(d, J=8.2 Hz, 2H) 7.93 (d, J=8.2 Hz, 2H).
[0180] Description 4
(2R)-2-Methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
hydrochloride
##STR00016##
[0182] To a solution of
1,1-dimethylethyl(3R)-3-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1--
piperazinecarboxylate (may be prepared as described in Description
3) (3.39 g, 8.30 mmol) in 1,4-dioxane (20 ml) was added HCl (4M in
1,4-dioxane) (10.4 ml, 41.5 mmol) and the resultant mixture stirred
under an atmosphere of Ar for 16 hours. A further 5 ml of 4M HCl in
dioxane was added and the mixture stirred at rt for 72 hours. The
mixture was concentrated to dryness and the residue triturated with
diethyl ether and the solid collected by filtration giving the
title compound (2.51 g) as a white powder.
[0183] m/z (API-ES) 309 [M+H].sup.+
[0184] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.16 (d,
J=7.0 Hz, 3H), 2.72-2.86 (m, 1H), 2.96 (dd, J=13.0, 4.28 Hz, 1H),
3.08-3.24 (m, 2H), 3.26-3.34 (m, 1H), 3.70-3.84 (m, 1H), 4.16-4.32
(m, 1H), 8.03 (d, J=8.4 Hz, 2H), 8.09 (d, J=8.3 Hz, 2H), 9.16 (br.
s. 2H).
[0185] Description 5
1,1-Dimethylethyl(3S)-3-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-p-
iperazinecarboxylate
##STR00017##
[0187] To a solution of
1,1-dimethylethyl(3S)-3-methyl-1-piperazinecarboxylate (2.05 g,
10.2 mmol) in DCM (50 ml) was added DIPEA (2.68 ml, 15.4 mmol) and
the mixture stirred at rt for 10 minutes before addition of
4-(trifluoromethyl)benzenesulfonyl chloride (3.00 g, 12.3 mmol) at
0.degree. C. The resultant mixture stirred under an atmosphere of
argon for 16 hours before addition of water (50 ml) and DCM (30
ml). The layers were separated using a hydrophobic frit and the
organic layers concentrated to dryness giving the title compound
(4.40 g) as a white solid.
[0188] m/z (API-ES) 309 [M+H-100].sup.+
[0189] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.03 (d, J=6.7
Hz, 3H), 1.43 (s, 9H), 2.59-3.33 (m, 3H), 3.43-4.35 (m, 4H), 7.77
(d, J=8.2 Hz, 2H), 7.93 (d, J=8.2 Hz, 2H).
[0190] Description 6
(2S)-2-Methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
hydrochloride
##STR00018##
[0192] To a solution of
1,1-dimethylethyl(3S)-3-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1--
piperazinecarboxylate (may be prepared as described in Description
5) (4.40 g, 10.8 mmol) in 1,4-dioxane (30 ml) was added HCl (4M in
1,4-dioxane) (5.39 ml, 21.6 mmol) and the mixture stirred at rt for
2 hours. A further portion of HCl (4M in 1,4-dioxane) (16.2 ml,
64.6 mmol) was then added and the mixture stirred for a further 16
hours. The volatiles were then removed in vacuo to give the title
compound (3.80 g) as a white solid.
[0193] m/z (API-ES) 309 [M+H].sup.+
[0194] 1H NMR (400 MHz, MeOH-d4) .delta. ppm 1.20 (d, J=7.1 Hz,
3H), 2.95-3.25 (m, 3H), 3.36-3.45 (m, 1H), 3.56-3.77 (m, 1H),
3.87-4.00 (m, 1H), 4.34-4.51 (m, 1H), 4.34-4.51 (m, 1H), 7.94 (d,
J=8.3 Hz, 2H), 8.08 (d, J=8.3 Hz, 2H).
[0195] Description 7
1,1-Dimethylethyl(2S)-4-[(4-bromo-2-methylphenyl)sulfonyl]-2-methyl-1-pipe-
razinecarboxylate
##STR00019##
[0197] To a solution of
1,1-dimethylethyl(2S)-2-methyl-1-piperazinecarboxylate (2.00 g,
9.99 mmol) and DIPEA (2.62 ml, 15.0 mmol) in dry DCM (25 ml) at
0.degree. C. under Ar was added 4-bromo-2-methylbenzenesulfonyl
chloride (2.96 g, 11.0 mmol) and the resulting yellow solution
allowed to warm to rt, then stirred at rt for 18 hours. Saturated
aq. NH.sub.4Cl (40 ml) was added, then the aqueous layer was
extracted with DCM (30 ml). The combined organic layers were passed
through a hydrophobic frit, then concentrated in vacuo to give a
yellow oil (5.01 g). Flash chromatography (silica; Flash 40M;
linear gradient (6-50%) EtOAc in isohexane) gave the title compound
as a very pale yellow oil
1,1-dimethylethyl(2S)-4-[(4-bromo-2-methylphenyl)sulfonyl]-2-methyl-1-pip-
erazinecarboxylate (3.52 g).
[0198] m/z (API-ES) 333 and 335, 1:1, [M+H-100].sup.+
[0199] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.19 (d, J=6.7
Hz, 3H), 1.44 (s, 9H), 2.59 (td, J=12.0, 3.4 Hz, 1H), 2.61 (s, 3H),
2.78 (dd, J=12.0, 3.8 Hz, 1H), 3.11 (td, J=12.0, 3.2 Hz, 1H), 3.44
(dt, J=12.0, 2.0 Hz, 1H), 3.59-3.65 (m, 1H), 3.93 (d, J=12.0 Hz,
1H), 4.33 (br. s., 1H), 7.45-7.51 (m, 2H), 7.72 (d, J=8.4 Hz,
1H).
[0200] Description 8
1,1-Dimethylethyl(2S)-4-[(4-cyano-2-methyl
phenyl)sulfonyl]-2-methyl-1-piperazinecarboxylate
##STR00020##
[0202] Ar was bubbled through a solution of
1,1-dimethylethyl(2S)-4-[(4-bromo-2-methylphenyl)sulfonyl]-2-methyl-1-pip-
erazinecarboxylate (may be prepared as described in Description 7)
(3.51 g, 8.10 mmol) in dry DMF (40 ml) for 0.5 hours, then
Zn(CN).sub.2 (0.523 g, 4.45 mmol), Pd.sub.2(dba).sub.3 (0.223 g,
0.243 mmol) and DPPF (0.269 g, 0.486 mmol) were added and the
resulting brown solution stirred at 120.degree. C. under Ar for 2.5
hours. The mixture was cooled to rt, concentrated in vacuo and the
residue partitioned between DCM (100 ml) and water (100 ml). The
aqueous layer was extracted with DCM (2.times.100 ml), then the
combined organic layers passed through a hydrophobic frit.
Concentration gave a brown residue (4.31 g). Flash chromatography
(silica; Flash 40M; linear gradient (6-50%) EtOAc in isohexane)
gave the title compound as a yellow solid (2.88 g).
[0203] m/z (API-ES) 280 [M+H-100].sup.+
[0204] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.20 (d, J=6.8
Hz, 3H), 1.44 (s, 9H), 2.65 (td, J=12.0, 3.4 Hz, 1H), 2.68 (s, 3H),
2.86 (dd, J=12.0, 4.2 Hz, 1H), 3.13 (td, J=12.0, 2.8 Hz, 1H), 3.49
(dt, J=12.0, 1.8 Hz, 1H), 3.63-3.69 (m, 1H), 3.95 (d, J=12.0 Hz,
1H), 4.35 (br. s., 1H), 7.61-7.65 (m, 2H), 7.96 (d, J=6.8 Hz,
1H).
[0205] Description 9
3-Methyl-4-{[(3S)-3-methyl-1-piperazinyl]sulfonyl}benzonitrile
##STR00021##
[0207] A solution of
1,1-dimethylethyl(2S)-4-[(4-cyano-2-methylphenyl)sulfonyl]-2-methyl-1-pip-
erazinecarboxylate (may be prepared as described in Description 8)
(2.88 g, 7.59 mmol) and TFA (10 ml, 130 mmol) in dry DCM (10 ml)
was stirred at rt for 1 hour, then concentrated in vacuo,
azetroping with toluene (25 ml) to give a brown oil. This was
partitioned between DCM (50 ml) and sat. aq. NaHCO.sub.3 (50 ml),
then the aq. layer extracted with DCM (50 ml). The combined organic
layers were passed through a hydrophobic frit and concentrated in
vacuo to give the title compound as a yellow oil (2.29 g).
[0208] m/z (API-ES) 280 [M+H].sup.+
[0209] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.06 (d, J=6.4
Hz, 3H), 2.33 (dd, J=11.6, 10.2 Hz, 1H), 2.67 (s, 3H), 2.69-2.75
(td, J=11.5, 3.1 Hz, 1H), 2.82-2.92 (m, 2H), 3.03 (dt, J=12.1, 2.6
Hz, 1H), 3.54-3.65 (m, 2H), 7.59-7.67 (m, 2H), 7.99 (d, J=8.6 Hz,
1H).
[0210] Description 10
(3R)-3-Methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
##STR00022##
[0212] To a solution of
1,1-dimethylethyl(2R)-2-methyl-1-piperazinecarboxylate (2.95 g,
14.7 mmol) in DCM (120 ml) was added DIPEA (5.40 ml, 30.9 mmol) and
then 4-(trifluoromethyl)benzenesulfonyl chloride (3.96 g, 16.2
mmol). The reaction mixture was stirred 2.5 hours at rt then washed
with water (250 ml), dried on a phase separation cartridge and
concentrated in vacuo. The obtained product was dissolved in
1,4-dioxane (60 ml) and treated with 4M HCl in 1,4-dioxane (18.4
ml, 73.6 mmol) overnight. The mixture was concentrated in vacuo
then dissolved in EtOAc (150 ml), washed with 2N aq. NaOH solution
(200 ml) then dried on a phase separation cartridge and
concentrated in vacuo. The product was then dissolved in EtOAc (100
ml) and extracted with 2M HCl (2.times.200 ml). 2M NaOH solution
were added to aqueous layer until basic pH then the product was
extracted with EtOAc (500 ml). The organic layer was dried on a
phase separation cartridge and concentrated in vacuo to give the
title compound (3.76 g).
[0213] m/z (API-ES) 309 [M+H].sup.+
[0214] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.05 (d, J=6.4
Hz, 3H), 1.93 (t, J=10.6 Hz, 1H), 2.31 (td, J=11.2, 3.4 Hz, 1H),
2.86-3.08 (m, 3H), 3.59-3.73 (m, 2H), 7.82 (d, J=8.3 Hz, 2H), 7.89
(d, J=8.3 Hz, 2H).
[0215] Description 11
1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine
##STR00023##
[0217] To a solution of 1,1-dimethylethyl 1-piperazinecarboxylate
(5 g, 26.8 mmol) in DCM (200 ml) was added DIPEA (9.85 ml, 56.4
mmol) and then 4-[(trifluoromethyl)oxy]benzenesulfonyl chloride
(4.55 ml, 26.8 mmol). The reaction mixture was stirred for 1.5
hours at rt. The reaction mixture was then evaporated to dryness in
vacuo. The residue was then dissolved in 1,4-dioxane (100 ml) and
4M HCl in 1,4-dioxane (50 ml) was added, along with a few drops of
distilled water. The reaction mixture was stirred for 3 hrs. Then
the reaction mixture was evaporated to dryness in vacuo. The
residue was dissolved in DCM (200 ml) and washed with 2M aq. NaOH
(50 ml, twice). The organic layer was dried over anhydrous
magnesium sulphate, the solid removed by filtration, and filtrate
evaporated to dryness in vacuo. The residue was not a solid so was
dissolved in ether and evaporated in vacuo to remove any remaining
solvent. The title compound (8.02 g) was obtained as a pale yellow
solid.
[0218] m/z (API-ES) 311 [M+H].sup.+
[0219] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 2.92-2.98 (m,
4H), 2.98-3.04 (m, 4H), 3.60-3.80 (m, 1H), 7.37 (d, J=8.9 Hz, 2H),
7.73-7.88 (d, J=8.9 Hz, 2H).
[0220] Description 12
(2R)-2-methyl-1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine
hydrochloride
##STR00024##
[0222] To a solution of
1,1-dimethylethyl(3R)-3-methyl-1-piperazinecarboxylate (605 mg,
2.56 mmol) in DCM (20 ml) was added DIPEA (1.12 ml, 6.39 mmol) and
then portionwise 4-[(trifluoromethyl)oxy]benzenesulfonyl chloride
(0.455 ml, 2.68 mmol) at room temp and the resultant mixture
stirred under Ar for 16 h. DCM (75 ml) and a 1M HCl solution (75
ml) were added, the layers were separated and the organic layer was
then washed with 2M NaOH solution (75 ml), the organic layers were
separated, dried (MgSO.sub.4) and concentrated to dryness.
1,1-Dimethylethyl(3R)-3-methyl-4-({4-[(trifluoromethyl)oxy]phenyl}sulfony-
l)-1-piperazinecarboxylate was isolated as a slowly crystallizing
white solid.
[0223] The solid was redissolved in 1,4-dioxane (4 ml), then HCl
was added (4M in 1,4-dioxane, 2.59 ml, 10.4 mmol) and the resultant
mixture stirred under an atmosphere of Ar for 2 h. After this time
a white precipitate had formed. MeOH (5 ml) was added and the
reaction stirred for a further 30 min. The reaction was
concentrated to dryness giving a white solid which was triturated
with Et.sub.2O and isolated by filtration under vacuum to leave the
title compound as a white solid (879 mg).
[0224] m/z (API-ES) 325 [M+H].sup.+
[0225] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.15 (d,
J=7.0 Hz, 3H), 2.80 (td, J=12.3, 3.7 Hz, 1H), 2.95 (dd, J=12.9, 4.4
Hz, 1H), 3.14 (t, J=14.3 Hz, 2H), 3.24-3.32 (m, 1H), 3.74 (d,
J=14.0 Hz, 1H), 4.16-4.25 (m, 1H), 7.63 (d, J=8.1 Hz, 2H), 8.01 (m,
J=8.8 Hz, 2H), 9.18 (br. s., 2H)
[0226] Description 13
(3S)-3-methyl-1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine
##STR00025##
[0228] To a solution of
1,1-dimethylethyl(2S)-2-methyl-1-piperazinecarboxylate (2.50 g,
12.5 mmol), DIPEA (4.58 ml, 26.2 mmol) and
4-[(trifluoromethyl)oxy]benzenesulfonyl chloride (3.25 g, 12.5
mmol) in DCM (200 ml) was stirred at room temperature overnight.
The mixture was washed with saturated aqueous NaHCO.sub.3, then
brine and concentrated under vacuum. The residue was redissolved in
1,4-dioxane (200 ml), then 4M HCl in dioxane (20 ml) and water (0.5
ml) were added and the mixture stirred overnight. The mixture was
concentrated under vacuum and applied to an SCX-2 cartridge (20 g)
washing with MeOH. The product was eluted with 0.5M NH.sub.3 in
MeOH; concentration left the title compound as a white solid (2.56
g).
[0229] m/z (API-ES) 325 [M+H].sup.+
[0230] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.04 (d,
J=6.4 Hz, 3H), 1.93 (t, J=10.6 Hz, 1H), 2.31 (td, J=11.2, 3.4 Hz,
1H), 2.86-3.07 (m, 3H), 3.58-3.68 (m, 2H), 7.37 (d, J=8.9 Hz, 2H),
7.77-7.84 (m, 2H)
[0231] Description 14
(3R)-3-methyl-1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine
##STR00026##
[0233] To a solution of
1,1-dimethylethyl(2R)-2-methyl-1-piperazinecarboxylate (2.00 g,
9.99 mmol) in DCM (200 ml) was added DIPEA (3.66 ml, 20.97 mmol)
and then 4-[(trifluoromethyl)oxy]benzenesulfonyl chloride (1.69 ml,
9.99 mmol) and the resulting mixture stirred at room temperature
for 90 min. The reaction mixture was then concentrated under vacuum
and redissolved in 1,4-dioxane (100 ml). A 4M solution of HCl in
1,4-dioxane (100 ml, 400 mmol) and a few drops of distilled water
were added and the mixture stirred for 3 h. The reaction mixture
was concentrated under vacuum, redissolved in DCM (200 ml) and
washed with 2M aqueous NaOH (2.times.50 ml). The organic layer was
dried (MgSO.sub.4), filtered and concentrated under vacuum. The
residue was dissolved in ether and concentrated under vacuum. The
oil was dissolved in MeOH (150 ml), applied to an SCX cartridge (50
g), which was washed with MeOH, DCM and MeOH again. The product was
eluted from the column with 2M ammonia in methanol, DCM and then 2M
ammonia in methanol; concentration under vacuum left the title
compound as a transparent yellow oil (2.92 g).
[0234] m/z (API-ES) 325 [M+H].sup.+
[0235] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.04 (d,
J=6.4 Hz, 3H), 1.93 (t, J=10.6 Hz, 1H), 2.31 (td, J=11.2, 3.5 Hz,
1H), 2.86-3.07 (m, 3H), 3.58-3.69 (m, 2H), 7.37 (d, J=8.1 Hz, 2H),
7.77-7.85 (m, 2H)
[0236] Description 15
Ethyl 6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
##STR00027##
[0238] A solution of (2Z)-3-(diethylamino)-2-fluoro-2-propenal (660
mg, 4.55 mmol--synthesized according to the literature procedure:
K. Funabiki, T. Ohtsuki, T. Ishihara and H. Yamanaka, Chem. Lett.,
1994, 1075-78.) and ethyl 3-amino-1H-pyrazole-4-carboxylate (1058
mg, 6.82 mmol) in acetic acid (5 ml) was heated at reflux for 1 h.
Additional ethyl 3-amino-1H-pyrazole-4-carboxylate (200 mg) was
added and the solution heated at reflux for a further 1 h.
Concentration under vacuum gave a pale yellow solid that was
suspended in DCM (25 ml) and 2M aqueous NaOH (25 ml). The organic
layers were isolated using a phase separator, then washed with 2M
aqueous HCl (25 ml), and isolated using a phase separator.
Concentration under vacuum gave a pale yellow solid (530 mg). Flash
chromatography (silica; Flash 12M; 2% [2M NH.sub.3 in MeOH] in DCM)
and concentration of the desired fractions gave the title compound
as a pale yellow solid (464 mg).
[0239] m/z (API-ES) 325 [M+H].sup.+
[0240] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.42 (t,
J=7.1 Hz, 3H), 4.45 (q, J=7.1 Hz, 2H), 8.60 (s, 1H), 8.72 (dd,
J=3.6, 2.7 Hz, 1H), 8.82 (d, J=2.7 Hz, 1H)
[0241] .sup.19F NMR (377 MHz, CHLOROFORM-d) .delta. ppm -149.6
(s)
[0242] Description 16
Lithium 6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate
##STR00028##
[0244] A suspension of ethyl
6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate (512 mg, 2.45 mmol)
and lithium hydroxide (61.5 mg, 2.57 mmol) in water (1 ml), MeOH (1
ml) and THF (1 ml) was irradiated to 100.degree. C. for 10 min in
the microwave. The reaction mixture was concentrated under vacuum,
azeotroping with toluene (10 ml) to leave the title compound as a
yellow powder (497 mg). This crude material was used directly in
the subsequent reactions.
[0245] Description 17
6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylic Acid
##STR00029##
[0247] A solution of ethyl 5-amino-1H-pyrazole-4-carboxylate (2 g,
12.89 mmol) and sodium hydroxide (19.34 mL, 38.7 mmol) (aqueous,
2M) in ethanol (30 mL) was heated at 80.degree. C. overnight. The
reaction mixture was concentrated in vacuo to give a crude mixture
of carboxylate and sodium hydroxide. The crude mixture was
dissolved in ethanol (4 mL) and acetic acid (14 mL, 245 mmol).
Whilst stirring the mixture, bromomalonaldehyde (1.947 g, 12.90
mmol) was added and then the mixture heated to 70.degree. C. for 1
h. The reaction mixture was allowed to cool and the precipitate was
filtered and washed with ethanol. The solid product was dried in a
vacuum oven to give the title compound (1.589 g).
[0248] m/z (API-ES) 242+244 (1:1) [M+H].sup.+
[0249] 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 8.58 (s, 1H),
8.88 (d, J=2.0 Hz, 1H), 9.76 (d, J=2.0 Hz, 1H), CO.sub.2H too broad
to observe.
[0250] Description 18
6-chloropyrazolo[1,5-a]pyrimidine-3-carboxylic Acid
##STR00030##
[0252] The preparation is similar to the one described in
Description 17.
[0253] m/z (API-ES) 198+200 (3:1) [M+H].sup.+
[0254] Description 19
1,1-dimethylethyl(2S)-4-{[2-bromo-5-(trifluoromethyl)phenyl]sulfonyl}-2-me-
thyl-1-piperazinecarboxylate
##STR00031##
[0256] To a solution of
1,1-dimethylethyl(2S)-2-methyl-1-piperazinecarboxylate (1.18 g,
4.98 mmol) in dichloromethane (40 ml) was added DIPEA (2.70 ml,
15.45 mmol) and then 2-bromo-5-(trifluoromethyl)benzenesulfonyl
chloride (1.613 g, 4.98 mmol). The reaction mixture was stirred for
1 h 20 min at room temperature, and then washed with water (50 ml),
dried on a phase separation cartridge and concentrated under vacuum
to give the crude title compound (2.5 g) used directly in the next
step.
[0257] m/z (API-ES) 387+389 (1:1) [(M-Boc)+H].sup.+
[0258] Description 20
1,1-dimethylethyl(2S)-2-methyl-4-{[2-methyl-5-(trifluoromethyl)phenyl]sulf-
onyl}-1-piperazinecarboxylate
##STR00032##
[0260]
1,1-dimethylethyl(2S)-4-{[2-bromo-5-(trifluoromethyl)phenyl]sulfony-
l}-2-methyl-1-piperazinecarboxylate (may be prepared as described
in Description 19) (2.5 g, 5.13 mmol), potassium carbonate (1.134
g, 8.21 mmol) in 1,4-dioxane (80 ml) were stirred for 5 min then
trimethylboroxin (1.142 ml, 8.21 mmol) and Pd(PPh.sub.3).sub.4
(0.593 g, 0.513 mmol) were added and the reaction mixture heated at
100.degree. C. for 1.5 h. Further trimethylboroxin (0.5 mL) was
added and the reaction heated for 30 min before it was allowed to
cool overnight.
[0261] The mixture was concentrated under vacuum then EtOAc (120
ml) added, washed with 200 ml of water, dried on a phase separation
cartridge and evaporated under vacuum. Crude material (2.8 g)
purified via Biotage (40+M silica column) using a gradient
EtOAc/i-Hex from 0/100 to 30/70. Desired fractions collected and
concentrated under vacuum to give the title compound (2.1 g).
[0262] m/z (API-ES) 323 [(M-Boc)+H].sup.+
[0263] Description 21
(3S)-3-methyl-1-{[2-methyl-5-(trifluoromethyl)phenyl]sulfonyl}piperazine
##STR00033##
[0265]
1,1-dimethylethyl(2S)-2-methyl-4-{[2-methyl-5-(trifluoromethyl)phen-
yl]sulfonyl}-1-piperazinecarboxylate (may be prepared as described
in Description 20) (2.1 g, 4.97 mmol) in 1,4-Dioxane (50 mL) was
treated by HCl 4M in dioxane (6.21 mL, 24.85 mmol). The reaction
mixture was stirred overnight at room temperature. LCMS showed a
1:1 ratio between starting material and expected product. HCl 4M in
dioxane (6.21 mL, 24.85 mmol) is added and the reaction mixture
stirred for 4 h. LCMS showed about 10% of remaining starting
material. HCl 4M in dioxane (6.21 mL, 24.85 mmol) is added and the
reaction mixture stirred for 1 h.
[0266] The reaction mixture was then concentrated under vacuum,
dissolved in EtOAc (100 ml) and extracted with 2N HCl (3.times.75
ml). 2N NaOH was added to the aqueous until basic then product
extracted with EtOAc, dried on a phase separation cartridge and
concentrated under vacuum to give the title compound (1.16 g).
[0267] m/z (API-ES) 323 [M+H].sup.+
[0268] Description 22
1,1-dimethylethyl
4-{[2-bromo-5-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinecarboxylate
##STR00034##
[0270] To a solution of 1,1-dimethylethyl 1-piperazinecarboxylate
(1 g, 5.37 mmol) in dichloromethane (40 ml) was added DIPEA (1.969
ml, 11.28 mmol) and then 2-bromo-5-(trifluoromethyl)benzenesulfonyl
chloride (1.737 g, 5.37 mmol). The mixture was stirred for 1 h 20
at room temperature before it was washed with water (50 ml), dried
on a phase separation cartridge and concentrated under vacuum.
[0271] Crude product was dissolved in MeOH and eluted through a 10
g SCX cartridge (elution with MeOH), concentrated under vacuum to
give the title compound (2.59 g).
[0272] m/z (API-ES) 373+375 (1:1) [(M-Boc)+H].sup.+
[0273] Description 23
1,1-dimethylethyl
4-{[2-methyl-5-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinecarboxylate
##STR00035##
[0275] 1,1-dimethylethyl
4-{[2-bromo-5-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinecarboxylate
(may be prepared as described in Description 22) (2.59 g, 5.47
mmol), potassium carbonate (1.513 g, 10.94 mmol) in 1,4-dioxane (80
ml) were stirred for 5 min then trimethylboroxin (1.523 ml, 10.94
mmol) and Pd(PPh.sub.3).sub.4 (0.632 g, 0.547 mmol) were added and
the reaction mixture heated at 100.degree. C. for 1.5 h. Further
trimethylboroxin (0.5 mL) was added and the mixture heated at
100.degree. C. for 30 min before allowing to cool overnight. The
mixture was concentrated under vacuum then EtOAc (150 ml) added,
washed with 200 ml of water, dried on a phase separation cartridge
and evaporated under vacuum. Crude material (3.1 g) purified via
Biotage (40+M silica column) using a gradient EtOAc/i-hex from
10/90 to 30/70. Desired fractions were collected and concentrated
under vacuum to give the title compound (2.1 g).
[0276] m/z (API-ES) 309 [(M-Boc)+H].sup.+
[0277] Description 24
1-{[2-methyl-5-(trifluoromethyl)phenyl]sulfonyl}piperazine
##STR00036##
[0279] 1,1-dimethylethyl
4-{[2-methyl-5-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinecarboxylate
(may be prepared as described in Description 23) (2.1 g, 5.14 mmol)
in 1,4-dioxane (50 mL) was treated by HCl 4M in dioxane (6.43 mL,
25.7 mmol). The reaction mixture was stirred overnight at room
temperature. LCMS showed a mixture of starting material and product
so 8 ml of HCl 4M in dioxane were added. LCMS after 2 h showed
still some starting material so 5 ml of HCl 4M in dioxane were
added. LCMS after 2 h showed traces of starting material.
[0280] The reaction mixture was concentrated under vacuum,
dissolved in EtOAc (70 ml) and extracted with 2N HCl (3.times.80
ml). 2N NaOH was added to the aqueous until basic, then product
extracted with EtOAc, dried on a phase separation cartridge and
concentrated under vacuum to give the title compound (1.38 g).
[0281] m/z (API-ES) 309 [M+H].sup.+
[0282] Description 25
(2S)-2-methyl-1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine
hydrochloride
##STR00037##
[0284] To a solution of
1,1-dimethylethyl(3S)-3-methyl-1-piperazinecarboxylate (500 mg,
2.497 mmol) in dichloromethane (20 ml) was added DIPEA (0.654 ml,
3.74 mmol) and then portionwise addition of
4-[(trifluoromethyl)oxy]benzenesulfonyl chloride (0.445 ml, 2.62
mmol) at room temperature and the resultant mixture stirred under
an atmosphere of argon for 16 hours. The reaction was worked up by
the addition of 1M HCl solution (75 mL) and dichloromethane (75
mL), the layers were separated and the organic layer was then
washed with 2M NaOH solution (75 mL). The organic phase was
separated, dried (MgSO.sub.4) and concentrated to dryness to give
1,1-dimethylethyl(3S)-3-methyl-4-({4-[(trifluoromethyl)oxy]phenyl}sulfony-
l)-1-piperazinecarboxylate (963 mg, 2.269 mmol). This was dissolved
in 1,4-dioxane (4 mL), HCl (4M in 1,4-Dioxane) (2.269 mL, 9.08
mmol) was added and the resultant mixture stirred under an
atmosphere of argon for 2 h. After this time a white precipitate
had formed so methanol (5 ml) was added to solubilize the
precipitate and the reaction stirred for a further 30 min. The
reaction was concentrated to dryness giving a white solid which was
triturated with Et.sub.2O and the solid isolated by filtration
under vacuum to give the title compound (585 mg).
[0285] m/z (API-ES) 325 [M+H].sup.+
[0286] Description 26
1,1-dimethylethyl(2S)-4-{[2-bromo-4-(trifluoromethyl)phenyl]sulfonyl}-2-me-
thyl-1-piperazinecarboxylate
##STR00038##
[0288] To a solution of
1,1-dimethylethyl(2S)-2-methyl-1-piperazinecarboxylate (1.20 g,
5.99 mmol) and DIPEA (5.45 ml, 31.2 mmol) in dry DCM (60 ml) at
0.degree. C. under Ar was added
2-bromo-4-(trifluoromethyl)benzenesulfonyl chloride (2.04 g, 6.29
mmol) and the resulting clear solution stirred at 0.degree. C. for
2 h. EtOAc (100 ml) and saturated aqueous NaHCO.sub.3 (100 ml) were
added, the layers separated, then the organic layer washed with 2M
aqueous HCl (100 ml) and passed through an hydrophobic frit. The
solvent was removed to leave the title compound (2.36 g).
[0289] m/z (API-ES) 387 and 389 [M+H-100].sup.+
[0290] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.02 (d,
J=6.7 Hz, 3H), 1.38 (s, 9H), 2.83 (td, J=12.3, 3.4 Hz, 1H), 3.00
(dd, J=12.6, 3.8 Hz, 1H), 3.07 (td, J=12.8, 3.0 Hz, 1H), 3.45 (d,
J=12.6 Hz, 1H), 3.73 (d, J=12.2 Hz, 1H), 3.81 (d, J=12.8 Hz, 1H),
4.15-4.24 (m, 1H), 7.99 (dd, J=8.2, 1.3 Hz, 1H), 8.18 (d, J=8.2 Hz,
1H), 8.30 (d, J=1.1 Hz, 1H)
[0291] Description 27
(3S)-3-methyl-1-{[2-methyl-4-(trifluoromethyl)phenyl]sulfonyl}piperazine
##STR00039##
[0293] A solution of
1,1-dimethylethyl(2S)-4-{[2-bromo-4-(trifluoromethyl)phenyl]sulfonyl}-2-m-
ethyl-1-piperazinecarboxylate (1.00 g, 2.05 mmol) (may be prepared
as described in Description 26), potassium carbonate (0.737 g, 5.34
mmol) in 1,4-dioxane (43 ml) were stirred for 5 min then
trimethylboroxin (0.743 ml, 5.34 mmol) and Pd(Ph.sub.3P).sub.4
(0.403 g, 0.349 mmol) were added and the reaction mixture heated at
100.degree. C. overnight. EtOAc (100 ml) was added then the mixture
washed aqueous sodium bicarbonate (100 ml), water (100 ml) and
concentrated under vacuum. Flash chromatography (silica; linear
gradient (0-20%) EtOAc in isohexane) gave
1,1-dimethylethyl(2S)-2-methyl-4-{[2-methyl-4-(trifluoromethyl)phenyl]sul-
fonyl}-1-piperazinecarboxylate (0.785 g).
[0294] This material was redissolved in DCM (10 ml) and 1,4-dioxane
(3 ml), then HCl in dioxane (5 eq) was added and the mixture
stirred at room temperature for 4 h. Aqueous sodium bicarbonate (20
ml) and EtOAc (20 ml) were added and the organic phase washed with
aqueous sodium bicarbonate (2.times.10 ml), brine (10 ml) and dried
using a hydrophobic frit. The solvent was removed under vacuum. The
residue was redissolved in DCM (10 ml) and 1,4-dioxane (3 ml), then
4M HCl in 1,4-dioxane (6.97 mL, 27.9 mmol) added. The mixture was
stirred at room temperature for 4 h. The solvent was evaporated
under vacuum to give the title compound (0.615 g) as the
hydrochloride salt.
[0295] m/z (API-ES) 323 [M+H].sup.+
[0296] .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.22 (d,
J=6.5 Hz, 3H), 2.66 (s, 3H), 2.77 (dd, J=12.8, 10.5 Hz, 1H),
2.93-3.11 (m, 2H), 3.29-3.39 (m, 2H), 3.63-3.75 (m, 2H), 7.84 (d,
J=8.3 Hz, 1H), 7.94 (s, 1H), 8.03 (d, J=8.3 Hz, 1H), 9.18 (br. s.,
2H)
Example 1
3-[(4-{[4-(Trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)carbonyl]pyrazol-
o[1,5-a]pyrimidine
##STR00040##
[0298] To a solution of
1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine (may be prepared
as described in Description 1) (200 mg, 0.680 mmol) in DMF (5 ml)
was added pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (111 mg,
0.680 mmol), HOBT.H.sub.2O (104 mg, 0.680 mmol) and HBTU (258 mg,
0.680 mmol). Finally DIPEA (0.356 ml, 2.039 mmol) was added and the
reaction mixture was stirred at rt for 20 hours. Solvent was
removed by evaporation, the crude partitioned between DCM (50 ml)
and saturated aq. sodium bicarbonate (50 ml), the DCM layer
collected, dried (hydrophobic frit) and evaporated. MDAP
purification yielded the title compound (317 mg) as an off-white
solid.
[0299] m/z (API-ES) 440 [M+H].sup.+
[0300] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 3.09-3.20 (m, 4H)
3.70-3.89 (m, 4H) 6.94 (dd, J=7.0, 4.0 Hz, 1H) 7.80 (d, J=8.5 Hz,
2H) 7.87 (d, J=8.5 Hz, 2H) 8.34 (s, 1H) 8.55 (dd, J=4.0, 2.0 Hz,
1H) 8.70 (dd, J=7.0, 2.0 Hz, 1H).
Example 2
3-[((2S)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)ca-
rbonyl]pyrazolo[1,5-a]pyrimidine
##STR00041##
[0302] To a solution of
(3S)-3-methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
(may be prepared as described in Description 2) (80.0 mg, 0.259
mmol) in DMF (5 ml) was added
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (42.3 mg, 0.259 mmol),
HOBT.H.sub.2O (39.7 mg, 0.259 mmol) and HBTU (98.0 mg, 0.259 mmol).
Finally DIPEA (0.136 ml, 0.778 mmol) was added and the reaction
mixture was stirred at room temperature for 3 hours. DMF was
evaporated in vacuo then 5 ml of DCM added and washed with
saturated aq. NaHCO.sub.3 solution (5 ml), dried on a phase
separation cartridge and evaporated in vacuo. The crude material
was dissolved in MeCN/DMSO 1:1 and purified by MDAP. The desired
fractions were collected and concentrated in vacuo to give the
title compound as a white solid (105 mg).
[0303] m/z (API-ES) 454 [M+H].sup.+
[0304] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.48 (d, J=6.8
Hz, 3H), 2.55 (td, J=11.8, 3.1 Hz, 1H), 2.68 (dd, J=11.6, 3.6 Hz,
1H), 3.41-3.57 (m, 1H), 3.64 (d, J=11.5 Hz, 1H), 3.81 (d, J=12.1
Hz, 1H), 4.04-4.42 (m, 1H), 4.63-4.96 (m, 1H), 6.96 (dd, J=7.0, 4.1
Hz, 1H), 7.84 (d, J=8.3 Hz, 2H), 7.90 (d, J=8.3 Hz, 2H), 8.38 (s,
1H), 8.58 (dd, J=4.1, 1.8 Hz, 1H), 8.73 (dd, J=7.0, 1.8 Hz,
1H).
Alternative preparation of
3-[((2S)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)c-
arbonyl]pyrazolo[1,5-a]pyrimidine: Example 2a
[0305] Pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (11.73 g, 71.9
mmol) was suspended in thionyl chloride (36 mL, 493 mmol) and
heated at 60.degree. C. for 2 h. Formation of the acyl chloride was
monitored as follows: a sample of the reaction mixture was
evaporated and added to MeOH and formation of the corresponding
methyl ester was detected by UPLC. Then thionyl chloride was
removed under reduced pressure to obtain
pyrazolo[1,5-a]pyrimidine-3-carbonyl chloride (14 g) as a yellow
solid.
[0306] (2S)-2-methylpiperazine (6 g, 59.9 mmol) was dissolved in
tetrahydrofuran (50 mL) and cooled down to 0.degree. C., aqueous
sodium hydroxide (3M, 39.9 mL, 120 mmol) was added and stirred for
10 min. Then 4-(trifluoromethyl)benzenesulfonyl chloride (16.12 g,
65.9 mmol) (dissolved in 50 ml of THF) was added drop-wise,
stirring the reaction mixture for 1 h. THF was removed under
reduced pressure, the aqueous phase was diluted with water (200 ml)
and extracted with DCM (2.times.300 ml). The organic layer was
concentrated under reduced pressure and the oily residue was
suspended with HCl 1M (200 ml) and washed with DCM in order to
extract impurities. NaOH 3M was added to the aqueous layer to reach
pH 10 then the mixture was diluted with THF (200 ml), the mixture
was cooled down to 0.degree. C.
[0307] The pyrazolo[1,5-a]pyrimidine-3-carbonyl chloride (14 g) was
suspended in THF (.about.80 ml) and added portion-wise to the above
mixture, maintaining the pH>9 by adding NaOH 3M, and then the
mixture was stirred overnight. THF was removed from the mixture
under reduced pressure and the resulting suspension was extracted
with DCM (2.times.300 ml). The organic layer was washed with HCl
0.1 M, dried over Na.sub.2SO.sub.4 and concentrated to dryness to
obtain the crude material (21.7 g) as foam. The crude material was
re-dissolved in DCM (100 ml) and evaporated to minimum volume to
obtain an oily residue, ethyl ether was added (80 ml) with
stirring. A solid crashed out from the solution and was recovered
by filtration, washing with ethyl ether before drying to give the
title compound (20.06 g).
[0308] m/z (API-ES) 454 [M+H].sup.+
[0309] 1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 1.48 (d, J=7.2 Hz,
3H), 2.56 (td, J=11.6, 3.2 Hz, 1H), 2.69 (dd, J=11.6, 3.6 Hz, 1H),
3.50 (m, 1H), 3.64 (d, J=11.6 Hz, 1H), 3.82 (d, J=11.2 Hz, 1H),
4.25 (m, 1H), 4.78 (m, 1H), 6.97 (dd, J=7.2, 4.0 Hz, 1H), 7.85 (d,
J=8.4 Hz, 2H), 7.91 (d, J=8.4 Hz, 2H), 8.39 (s, 1H), 8.59 (dd,
J=4.0, 1.6 Hz, 1H), 8.73 (dd, J=7.2, 1.6 Hz, 1H).
Example 3
3-[((3R)-3-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)ca-
rbonyl]pyrazolo[1,5-a]pyrimidine
##STR00042##
[0311] To a solution of pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
(56.8 mg, 0.348 mmol) in DCM (2.0 ml) was added HATU (132 mg, 0.348
mmol) and DIPEA (0.152 ml, 0.870 mmol) and the mixture stirred for
15 minutes before addition of
(2R)-2-methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
hydrochloride (may be prepared as described in Description 4) (100
mg, 0.290 mmol) in DMF (2 ml). The resultant mixture was stirred
for 72 hours at rt. Water (20 ml) and EtOAc (20 ml) were added and
the mixture was filtered through a sinter to remove precipitate.
The collected filtrate was separated and the aqueous layer was then
re-extracted with EtOAc (15 ml), the organic layers were combined
and washed with saturated brine solution (20 ml). The organic
layers were then separated, dried (MgSO.sub.4) and concentrated to
dryness. The crude material was then dissolved in DMSO and purified
by MDAP. The collected fractions were concentrated in vacuo to give
the title compound (85.8 mg) as a white solid.
[0312] MS ES+ m/z 454 (M+H).sup.+
[0313] 1H NMR (400 MHz, CHLOROFORM-d) rotameric mixture .delta. ppm
1.13 (br. s., 3H), 2.73-3.84 (m, 4H), 3.86-5.00 (m, 3H), 6.97 (dd,
J=7.0, 4.1 Hz, 1H), 7.80 (d, J=8.2 Hz, 2H), 7.97 (d, J=8.1 Hz, 2H),
8.41 (s, 1H), 8.61 (dd, J=4.1, 1.81 Hz, 1H), 8.74 (dd, J=7.0, 1.8
Hz, 1H).
Example 4
3-[((3S)-3-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)ca-
rbonyl]pyrazolo[1,5-a]pyrimidine
##STR00043##
[0315] To a solution of
(2S)-2-methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
hydrochloride (may be prepared as described in Description 6) (100
mg, 0.270 mmol) in THF (5 ml) was added HOBT.H.sub.2O (41.3 mg,
0.270 mmol), HBTU (102 mg, 0.270 mmol) and
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (48.4 mg, 0.297 mmol).
The mixture was stirred for 5 minutes before addition of DIPEA
(0.118 ml, 0.674 mmol) and the resultant solution stirred at rt for
16 hours. The mixture was then concentrated and the residue
partitioned between DCM (10 ml) and water (10 ml), the layers were
separated using a hydrophobic frit and the organic layers
concentrated to dryness and the crude material dissolved in DMSO
and purified by MDAP to give the title compound (85.3 mg) as a
white solid.
[0316] MS ES+ m/z 454 (M+H).sup.+
[0317] 1H NMR (400 MHz, CHLOROFORM-d) rotamic mixture .delta. ppm
0.86-1.28 (m, 3H), 3.49 (s, 3H), 3.63-3.84 (m, 1H), 3.86-4.94 (m,
3H), 6.97 (dd, J=7.0, 4.1 Hz, 1H), 7.80 (d, J=8.3 Hz, 2H), 7.97 (d,
J=8.2 Hz, 2H), 8.41 (s, 1H), 8.61 (dd, J=4.1, 1.8 Hz, 1H), 8.73
(dd, J=7.0, 1.8 Hz, 1H).
Example 5
3-Methyl-4-{[(3S)-3-methyl-4-(pyrazolo[1,5-a]pyrimidin-3-ylcarbonyl)-1-pip-
erazinyl]sulfonyl}benzonitrile
##STR00044##
[0319] To a solution of
3-methyl-4-{[(3S)-3-methyl-1-piperazinyl]sulfonyl}benzonitrile (may
be prepared as described in Description 9) (75 mg, 0.268 mmol),
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (48.2 mg, 0.295 mmol)
and DIPEA (0.070 ml, 0.403 mmol) in dry DMF (3 ml) at rt under
argon was added HATU (122 mg, 0.322 mmol) and the resulting yellow
solution stirred at rt for 1 hour. Concentration in vacuo gave a
yellow oil, that was purified by MDAP; concentration of the desired
fractions gave a clear film (80.2 mg). Flash chromatography
(silica; Flash 12S; linear gradient (1-8%) [2M NH.sub.3 in MeOH] in
DCM) gave the title compound as a clear film (44.9 mg), that became
a white solid on standing in vacuo (1 mbar) for 1 h.
[0320] m/z (API-ES) 425 [M+H].sup.+
[0321] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.38 (d, J=6.9
Hz, 3H), 2.70 (s, 3H), 2.95 (td, J=11.9, 2.4 Hz, 1H), 3.11 (dd,
J=12.1, 3.5 Hz, 1H), 3.37-3.50 (m, 1H), 3.60 (d, J=12.1 Hz, 1H),
3.74 (d, J=11.9 Hz, 1H), 4.23 (br. s., 1H), 4.77 (br. s., 1H), 6.97
(dd, J=7.0, 4.1 Hz, 1H), 7.61-7.67 (m, 2H), 7.98 (d, J=8.7 Hz, 1H),
8.41 (s, 1H), 8.60 (dd, J=4.1, 1.8 Hz, 1H), 8.73 (dd, J=7.0, 1.8
Hz, 1H).
Example 6
6-Methyl-3-[(4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)carbony-
l]pyrazolo[1,5-a]pyrimidine
##STR00045##
[0323] To a solution of
1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine (may be prepared
as described in Description 1) (100 mg, 0.340 mmol) in DMF (5 ml)
was added HOBT.H.sub.2O (52.0 mg, 0.340 mmol), HBTU (129 mg, 0.340
mmol), 6-methylpyrazolo[1,5-a]pyrimidine-3-carboxylic acid (60.2
mg, 0.340 mmol) and DIPEA (0.178 ml, 1.019 mmol), in a 2.0-5.0 ml
microwave vial. The reaction mixture was stirred for 3 hours at rt.
The reaction mixture was transferred to a 100 ml round bottom flask
and was reduced to dryness in vacuo. The residue was dissolved in
DCM (50 ml) and was transferred to a separating funnel then washed
with saturated aq. NaHCO.sub.3 solution (5 ml), twice. The organic
layer was collected and dried with dried magnesium sulfate. The
solid was removed by filtration and the filtrate collected in a 250
ml round bottom flask and reduced to dryness in vacuo. The residue
was then dissolved in 1.8 ml (1:1) MeCN/DMSO and purified by MDAP
in 2 batches. The fractions containing desired product were
combined in a 250 ml round bottom flask and reduced in vacuo to
yield the title compound (79 mg).
[0324] m/z (API-ES) 454 [M+H].sup.+
[0325] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 2.43 (d, J=0.9
Hz, 3H), 3.04-3.29 (m, 4H), 3.80-3.90 (m, 4H), 7.78-7.98 (m, 4H),
8.32 (s, 1H), 8.40-8.58 (m, 2H).
Example 7
3-[((2R)-2-Methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)ca-
rbonyl]pyrazolo[1,5-a]pyrimidine
##STR00046##
[0327] To a solution of
(3R)-3-methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
(may be prepared as described in Description 10) (80.0 mg, 0.259
mmol) in DMF (4 ml) was added
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (42.3 mg, 0.259 mmol),
HOBT.H.sub.2O (39.7 mg, 0.259 mmol), HBTU (98 mg, 0.259 mmol) and
DIPEA (0.136 ml, 0.778 mmol) and the reaction mixture was stirred
at rt for 1.5 hours. The DMF was evaporated in vacuo then DCM (5
ml) was added and the whole washed with saturated aq. NaHCO.sub.3
solution (5 ml), dried on a phase separation cartridge and
evaporated in vacuo. The crude material was dissolved in MeCN/DMSO
1:1 and purified by MDAP to give the title compound (100 mg).
[0328] m/z (API-ES) 454 [M+H].sup.+
[0329] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.47 (d, J=6.8
Hz, 3H), 2.55 (td, J=11.7, 3.2 Hz, 1H), 2.68 (dd, J=11.5, 3.6 Hz,
1H), 3.40-3.57 (m, 1H), 3.63 (d, J=11.4 Hz, 1H), 3.81 (d, J=10.7
Hz, 1H), 4.10-4.39 (m, 1H), 4.62-4.95 (m, 1H) 6.96 (dd, J=7.0, 4.1
Hz, 1H), 7.84 (d, J=8.3 Hz, 2H), 7.90 (d, J=8.3 Hz, 2H), 8.38 (s,
1H), 8.58 (dd, J=4.1, 1.8 Hz, 1H), 8.73 (dd, J=7.0, 1.8 Hz,
1H).
Example 8
3-{[4-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)-1-piperazinyl]carbonyl}py-
razolo[1,5-a]pyrimidine
##STR00047##
[0331] To a solution of
1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine (may be
prepared as described in Description 11) (300 mg, 0.967 mmol) in
DMF (15 ml) was added pyrazolo[1,5-a]pyrimidine-3-carboxylic acid
(158 mg, 0.967 mmol), HOBT.H.sub.2O (148 mg, 0.967 mmol) and HBTU
(368 mg, 0.967 mmol). Finally DIPEA (0.507 ml, 2.90 mmol) was added
and the reaction mixture was stirred at rt for 20 hours. Solvent
was removed by evaporation, the crude material partitioned between
DCM (50 ml) and saturated sodium bicarbonate (50 ml), the DCM layer
collected, washed with 2N HCl aq. (50 ml), dried (hydrophobic frit)
and evaporated. The crude was purified by MDAP. MDAP fractions were
evaporated and the crude material partitioned between DCM (20 ml)
and sat. sodium bicarbonate solution (20 ml). The DCM layer was
collected and dried (hydrophobic frit) and evaporated to give the
title compound (308 mg).
[0332] m/z (API-ES) 456 [M+H].sup.+
[0333] 1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 3.16 (d, 4H),
3.84 (br. s., 4H), 6.96 (dd, J=7.0, 4.1 Hz, 1H), 7.32-7.45 (m, 2H),
7.76-7.88 (m, 2H), 8.38 (s, 1H), 8.58 (dd, J=4.1, 1.8 Hz, 1H), 8.72
(dd, J=7.0, 1.8 Hz, 1H).
Example 9
6-Fluoro-3-[((2S)-2-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piper-
azinyl)carbonyl]pyrazolo[1,5-a]pyrimidine
##STR00048##
[0335] The lithium salt of
6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate (117 mg, 0.63 mmol)
(may be prepared as described in Description 16) was weighed into a
vial with HATU (247 mg, 0.65 mmol), suspended in DMF (2 ml) and
treated with DIPEA (0.170 ml, 0.97 mmol). This mixture was stirred
about 15 min at ambient temperature.
(3S)-3-Methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
(may be prepared as described in Description 2) (100 mg, 0.32 mmol)
was added and stirring was continued for 3 h at ambient
temperature. The reaction mixture was partitioned between DCM and
sat. aq. NaHCO.sub.3 solution (10 ml each). The layers were
separated (hydrophobic frit) and the aqueous layer was washed with
further DCM (2.times.5 ml). The combined organic layers were
concentrated to leave an orange gum. Purification by MDAP gave the
title compound (112 mg, 0.24 mmol, 73% yield) as an orange
solid.
[0336] m/z (API-ES) 472 [M+H].sup.+
[0337] 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.28 (d, J=6 Hz,
3H), 2.38-2.48 (m, 1H), 2.54 (dd, J=11, 3 Hz, 1H), 3.28-3.40 (m,
1H, obscured by water), 3.47-3.55 (m, 1H), 3.64-3.73 (m, 1H),
3.90-4.15 (m, 1H), 4.40-4.70 (m, 1H), 7.97 (d, J=8 Hz, 2H), 8.06
(d, J=8 Hz, 2H), 8.40 (s, 1H), 8.90 (d, J=3 Hz, 1H), 9.64 (dd, J=5,
3 Hz, 1H).
Example 10
4-({(3S)-4-[(6-fluoropyrazolo[1,5-a]pyrimidin-3-yl)carbonyl]-3-methyl-1-pi-
perazinyl}sulfonyl)-3-methylbenzonitrile
##STR00049##
[0339] To a solution of
3-methyl-4-{[(3S)-3-methyl-1-piperazinyl]sulfonyl}benzonitrile (may
be prepared as described in Description 9) (100 mg, 0.317 mmol),
lithium 6-fluoropyrazolo[1,5-a]pyrimidine-3-carboxylate (may be
prepared as described in Description 16) (65.5 mg, 0.348 mmol),
HOBT (58.2 mg, 0.380 mmol) and Et.sub.3N (0.110 ml, 0.792 mmol) in
dry DMF (50 ml) at rt under Ar was added HBTU (144 mg, 0.380 mmol)
and the resulting dark brown solution stirred at rt for 16 h. The
mixture was concentrated under vacuum to leave a black oil.
Purification by MDAP and concentration of the desired fractions
gave a yellow film (109 mg). Flash chromatography (silica; Flash
12M; 3:1, EtOAc:isohexane) gave the title compound as a white foam
(57.1 mg), that became a white solid on trituration with ether.
[0340] m/z (API-ES) 443 [M+H].sup.+
[0341] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.38 (d,
J=6.9 Hz, 3H), 2.69 (s, 3H), 2.91 (td, J=12.1, 3.1 Hz, 1H), 3.08
(dd, J=12.2, 3.6 Hz, 1H), 3.35-3.52 (m, 1H), 3.60 (d, J=12.2 Hz,
1H), 3.74 (d, J=12.1 Hz, 1H), 4.12 (br. s., 1H), 4.72 (br. s., 1H),
7.61-7.67 (m, 2H), 7.98 (d, J=8.7 Hz, 1H), 8.40 (s, 1H), 8.65 (d,
J=2.6 Hz, 1H), 8.69 (dd, J=3.5, 2.7 Hz, 1H)
Example 11
3-{[(2S)-2-methyl-4-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)-1-piperazin-
yl]carbonyl}pyrazolo[1,5-a]pyrimidine
##STR00050##
[0343] To a solution of
(3S)-3-methyl-1-({4-[(trifluoromethyl)oxy]phenyl}sulfonyl)piperazine
(may be prepared as described in Description 13) (300 mg, 0.925
mmol), HOBt (142 mg, 0.925 mmol), HBTU (351 mg, 0.925 mmol) and
pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (151 mg, 0.925 mmol) in
DMF (50 ml) was added DIPEA (0.485 ml, 2.78 mmol) and the resulting
mixture stirred at room temperature overnight, then concentrated
under vacuum. The residue was redissolved in EtOAc, washed with
aqueous NaHCO.sub.3 and brine, then concentrated under vacuum.
Flash chromatography (silica; EtOAc) and concentration of the
desired fractions gave the title compound (157 mg).
[0344] m/z (API-ES) 470 [M+H].sup.+
[0345] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.45 (d,
J=6.8 Hz, 3H), 2.53 (td, J=11.7, 3.3 Hz, 1H), 2.66 (dd, J=11.5, 3.6
Hz, 1H), 3.36-3.54 (m, 1H), 3.60 (d, J=11.3 Hz, 1H), 3.77 (d,
J=10.9 Hz, 1H), 4.30 (br. s., 1H), 4.76 (br. s., 1H), 6.95 (dd,
J=7.0, 4.1 Hz, 1H), 7.37 (dd, J=8.9, 0.9 Hz, 2H), 7.77-7.84 (m,
2H), 8.35 (s, 1H), 8.57 (dd, J=4.1, 1.8 Hz, 1H), 8.72 (dd, J=7.0,
1.8 Hz, 1H)
Example 12
6-bromo-3-[((2S)-2-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-pipera-
zinyl)carbonyl]pyrazolo[1,5-a]pyrimidine
##STR00051##
[0347] To a solution of
6-bromopyrazolo[1,5-a]pyrimidine-3-carboxylic acid (may be prepared
as described in Description 17) (471 mg, 1.946 mmol) in
N,N-Dimethylformamide (10 mL) under a flush of argon was added HATU
(740 mg, 1.946 mmol) and the reaction stirred for 10 minutes.
(3S)-3-methyl-1-{[4-(trifluoromethyl)phenyl]sulfonyl}piperazine
(may be prepared as described in Description 2) (600 mg, 1.946
mmol) was then added followed by N,N-diisopropylethylamine (0.680
mL, 3.89 mmol) and the reaction stirred for 18 h. The reaction
mixture was partitioned between ethyl acetate (60 mL) and water (40
mL). The organic layer was washed with further water (20 mL) and
brine (20 mL) before it was dried (MgSO.sub.4), filtered and the
solvent removed in vacuo. The crude material was purified by silica
chromatography, 40 M cartridge, eluting 0-75% EtOAC in isohexane
and the solvent evaporated to give the title compound (587 mg) as a
white solid.
[0348] m/z (API-ES) 532+534 (1:1) [M+H].sup.+
[0349] 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.28 (d, J=7.2
Hz, 3H), 2.35-2.55 (m, 3H), 3.51 (m, 1H), 3.68 (m, 1H), 4.02 (m,
1H), 4.57 (m, 1H), 7.97 (d, J=8.4 Hz, 2H), 8.06 (d, J=8.4 Hz, 2H),
8.37 (s, 1H), 8.73 (d, J=2.4 Hz, 1H), 9.69 (d, J=2.4 Hz, 1H).
Example 13
3-[((2S)-2-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piperazinyl)ca-
rbonyl]pyrazolo[1,5-a]pyrimidine-6-carbonitrile
##STR00052##
[0351] To a mixture of
6-bromo-3-[((2S)-2-methyl-4-{[4-(trifluoromethyl)phenyl]sulfonyl}-1-piper-
azinyl)carbonyl]pyrazolo[1,5-a]pyrimidine (may be prepared as
described in Example 12) (300 mg, 0.564 mmol) and
1,1'-bis(diphenylphosphino)ferrocene (39.1 mg, 0.070 mmol) under
argon in dry N,N-dimethylformamide (6 mL) was added zinc cyanide
(86 mg, 0.733 mmol) followed by
tris(dibenzylideneacetone)dipalladium(0) (32.0 mg, 0.035 mmol) and
the mixture heated to 95.degree. C. for 2 h. The mixture was
partitioned between ethyl acetate (30 mL) and aqueous sodium
bicarbonate (20 mL). The organic phase was washed with further
sodium bicarbonate (10 mL), water (10 mL) and brine (10 mL) before
it was dried (MgSO.sub.4), filtered and the solvent removed in
vacuo. Half the crude material was purified by MDAP and the solvent
evaporated to give the title compound (67 mg) as a yellow
solid.
[0352] m/z (API-ES) 479 [M+H].sup.+
[0353] 1H NMR (400 MHz, DMSO-d.sub.6) .delta. ppm 1.28 (d, J=6.8
Hz, 3H), 2.4-2.6 and 3.4-4.7 (m, 5H), 7.97 (d, J=8.0 Hz, 2H), 8.06
(d, J=8.0 Hz, 2H), 8.59 (s, 1H), 8.91 (d, J=2.0 Hz, 1H), 10.07 (d,
J=2.0 Hz, 1H).
Examples 14 to 24
[0354] The compounds of Table 1 were prepared in a similar manner
as the compound of Example 1 using the corresponding reactants.
TABLE-US-00001 TABLE I Example m/z (API- no. Name Structure ES) [M
+ H].sup.+ 14 5,7-dimethyl-3-[(4-{[4-
(trifluoromethyl)phenyl]sulfonyl}-1-
piperazinyl)carbonyl]pyrazolo[1,5-a] pyrimidine ##STR00053## 468 15
3-{[(3S)-3-methyl-4-({4- [(trifluoromethyl)oxy]phenyl} sulfonyl)-1-
piperazinyl]carbonyl}pyrazolo[1,5-a] pyrimidine ##STR00054## 470 16
4-({(3S)-4-[(6- chloropyrazolo[1,5- a]pyrimidin-3-yl)carbonyl]-3-
methyl-1- piperazinyl}sulfonyl)-3- methylbenzonitrile ##STR00055##
459 + 461 (3:1) 17 6-chloro-3-[((2S)-2-methyl-4- {[4-
(trifluoromethyl)phenyl]sulfonyl}-1-
piperazinyl)carbonyl]pyrazolo[1,5-a] pyrimidine ##STR00056## 488 +
490 (3:1) 18 3-[((2S)-2-methyl-4-{[2- methyl-5-
(trifluoromethyl)phenyl]sulfonyl}-1-
piperazinyl)carbonyl]pyrazolo[1,5-a] pyrimidine ##STR00057## 468 19
3-[(4-{[2-methyl-5- (trifluoromethyl)phenyl]sulfonyl}-1-
piperazinyl)carbonyl]pyrazolo[1,5-a] pyrimidine ##STR00058## 454 20
2-methyl-3-[((2S)-2-methyl- 4-{[4-
(trifluoromethyl)phenyl]sulfonyl}-1-
piperazinyl)carbonyl]pyrazolo[1,5-a] pyrimidine ##STR00059## 468 21
3-{[(3R)-3-methyl-4-({4- [(trifluoromethyl)oxy]phenyl} sulfonyl)-1-
piperazinyl]carbonyl}pyrazolo[1,5-a] pyrimidine ##STR00060## 470 22
3-{[(2R)-2-methyl-4-({4- [(trifluoromethyl)oxy]phenyl} sulfonyl)-1-
piperazinyl]carbonyl}pyrazolo[1,5-a] pyrimidine ##STR00061## 470 23
3-methyl-4-({(3S)-3-methyl- 4-[(6-methylpyrazolo[1,5-
a]pyrimidin-3-yl)carbonyl]-1- piperazinyl}sulfonyl)benzonitrile
##STR00062## 439 24 3-methyl-4-({(3S)-3-methyl-
4-[(2-methylpyrazolo[1,5- a]pyrimidin-3-yl)carbonyl]-1-
piperazinyl}sulfonyl)benzonitrile ##STR00063## 439
Example 25
3-[((2S)-2-methyl-4-{[2-methyl-4-(trifluoromethyl)phenyl]sulfonyl}-1-piper-
azinyl)carbonyl]pyrazolo[1,5-a]pyrimidine
##STR00064##
[0356] To a solution of
(3S)-3-methyl-1-{[2-methyl-4-(trifluoromethyl)phenyl]sulfonyl}piperazine
(100 mg, 0.310 mmol) (may be prepared as described in Description
27), pyrazolo[1,5-a]pyrimidine-3-carboxylic acid (55.7 mg, 0.341
mmol), HOBt (52.3 mg, 0.341 mmol) and DIPEA (0.081 ml, 0.465 mmol)
in dry DMF (5 ml) at rt was added HBTU (129 mg, 0.341 mmol) and the
resulting orange solution stirred at rt for 45 min. Concentration
under vacuum left an orange oil. Purification by MDAP and
concentration of the desired fractions gave the title compound (108
mg) as a clear film that became a white solid on trituration with
ether.
[0357] m/z (API-ES) 468 [M+H].sup.+
[0358] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm 1.39 (d,
J=6.9 Hz, 3H), 2.73 (s, 3H), 2.94 (td, J=11.9, 2.7 Hz, 1H), 3.10
(dd, J=12.1, 3.6 Hz, 1H), 3.36-3.54 (m, 1H), 3.61 (d, J=12.1 Hz,
1H), 3.75 (d, J=12.0 Hz, 1H), 4.22 (br. s., 1H), 4.77 (br. s., 1H),
6.97 (dd, J=7.0, 4.1 Hz, 1H), 7.57-7.66 (m, 2H), 7.96-8.06 (m, 1H),
8.42 (s, 1H), 8.61 (dd, J=4.1, 1.8 Hz, 1H), 8.74 (dd, J=7.0, 1.8
Hz, 1H)
[0359] Equipment:
[0360] Mass-Directed Automated HPLC/Mass-Directed Automated
Preparation (MDAP)
[0361] Where indicated in the above Examples, purification by
mass-directed automated HPLC was carried out using the following
apparatus and conditions:
[0362] Hardware
[0363] Waters 2525 Binary Gradient Module
[0364] Waters 515 Makeup Pump
[0365] Waters Pump Control Module
[0366] Waters 2767 Inject Collect
[0367] Waters Column Fluidics Manager
[0368] Waters 2996 Photodiode Array Detector
[0369] Waters ZQ Mass Spectrometer
[0370] Gilson 202 fraction collector
[0371] Gilson Aspec waste collector
[0372] Software
[0373] Waters MassLynx version 4 SP2
[0374] Column
[0375] The columns used are Waters Atlantis, the dimensions of
which are 19 mm.times.100 mm (small scale) and 30 mm.times.100 mm
(large scale). The stationary phase particle size is 5 .mu.m.
[0376] Solvents
[0377] A: Aqueous solvent=Water+0.1% Formic Acid
[0378] B: Organic solvent=Acetonitrile+0.1% Formic Acid
[0379] Make up solvent=Methanol:Water 80:20
[0380] Needle rinse solvent=Methanol
[0381] Methods
[0382] There are five methods used depending on the analytical
retention time of the compound of interest. They have a 13.5-minute
runtime, which comprises of a 10-minute gradient followed by a 3.5
minute column flush and re-equilibration step.
[0383] Large/Small Scale 1.0-1.5=5-30% B
[0384] Large/Small Scale 1.5-2.2=15-55% B
[0385] Large/Small Scale 2.2-2.9=30-85% B
[0386] Large/Small Scale 2.9-3.6=50-99% B
[0387] Large/Small Scale 3.6-5.0=80-99% B (in 6 minutes followed by
7.5 minutes flush and re-equilibration)
[0388] Flow Rate
[0389] All of the above methods have a flow rate of either 20
mls/min (Small Scale) or 40 mls/min (Large Scale).
[0390] Liquid Chromatography/Mass Spectrometry
[0391] Analysis of the above compounds by Liquid
Chromatography/Mass Spectrometry (LC/MS) was carried out using the
following apparatus and conditions:
[0392] Hardware
[0393] Waters Acquity Binary Solvent Manager
[0394] Waters Acquity Sample Manager
[0395] Waters Acquity PDA
[0396] Waters ZQ Mass Spectrometer
[0397] Sedere Sedex 75
[0398] Software
[0399] Waters MassLynx version 4.1
[0400] Column
[0401] The column used is a Waters Acquity BEH UPLC C18, the
dimensions of which are 2.1 mm.times.50 mm. The stationary phase
particle size is 1.7 .mu.m.
[0402] Solvents
[0403] A: Aqueous solvent=Water+0.05% Formic Acid
[0404] B: Organic solvent=Acetonitrile+0.05% Formic Acid
[0405] Weak Wash=1:1 Methanol:Water
[0406] Strong Wash=Water
[0407] Method
[0408] The generic method used has a 2 minute runtime.
TABLE-US-00002 Time/min % B 0 3 0.1 3 1.5 97 1.9 97 2.0 3
[0409] The above method has a flow rate of 1 ml/min.
[0410] The injection volume for the generic method is 0.5 ul
[0411] The column temperature is 40.degree. C.
[0412] The UV detection range is from 220 to 330 nm
[0413] Biotage SP4.RTM.
[0414] Biotage-SP4.RTM. is an automated purification system. It
uses preloaded silica gel columns. The user applies their material
to the top of the column and chooses solvents, gradients, flow
rates, column size, collection method and eluting volumes.
[0415] Phase Separators (Hydrophobic Frit)
[0416] Phase separators are a range of ISOLUTE.RTM. columns fitted
with an optimized frit material that easily separates aqueous phase
from chlorinated solvents under gravity.
[0417] SCX--Strong Cation Exchange Cartridge
[0418] Where indicated in the compounds, an SCX cartridge was used
as part of the compound purification process. Typically an ISOLUTE
SCX-2 cartridge was used. ISOLUTE SCX-2 is a silica-based sorbent
with a chemically bonded propylsulfonic acid functional group.
[0419] ISOLUTE SCX-2 Chemical Data
[0420] Base Material: Silica, 50 .mu.m
[0421] Functional Group: Propylsulfonic acid
[0422] Capacity: 0.6 meq/g
[0423] Counter Ion: Proton
[0424] Pharmacological Data
[0425] Compounds of the invention may be tested for in vitro
biological activity in the hCa.sub.V2.2 assay in accordance with
the following studies:
[0426] Methods
[0427] Cell Biology
[0428] Stable cell lines expressing the human Ca.sub.V2.2 .alpha.
(.alpha.1.sub.B) subunit, along with the human .beta.3 and
.alpha.2.delta.1 auxillary subunits were created following
sequential transfection and selection of human embryonic kidney
(HEK293) cells. HEK293 cells were cultured in Dulbecco's modified
Eagles media/F12 media (Invitrogen, Cat #041-95750V) containing 10%
fetal bovine serum, with added L-glutamine (2 mM; Invitrogen, Cat
#25030-024) and non-essential amino acids (5%; Invitrogen, Cat
#11140-035). Initially HEK293 cells were transfected with two
plasmid vectors for expression of the hCa.sub.V2.2 .alpha. subunit
(pCIN5-hCa.sub.V2.2 which carries a neomycin resistance marker) and
the hCa.sub.V .beta.3 subunit (pCIH-hCa.sub.V .beta.3 which carries
a hygromycin resistance marker). Clonal cell lines were isolated
following selection in media supplemented with 0.4 mg ml.sup.-1
Geneticin G418 (Invitrogen, Cat #10131-027) and 0.1 mg ml.sup.-1
hygromycin (Invitrogen, Cat #10687-010). These clonal cell lines
were assessed for Ca.sub.v2.2 .alpha./.beta.3-mediated current
expression using the IonWorks planar array electrophysiology
technology (described below). A clonal line was identified that
gave a reasonable level of functional Ca.sub.v2.2 .alpha./.beta.3
current expression. This cell line was transfected with a plasmid
vector for expression of the human .alpha.2.delta.1 subunit
(pCIP-.alpha.2.delta.1 which carries a puromycin resistance marker)
and clonal cell lines isolated following selection in media
containing 0.62 .mu.g ml.sup.-1 puromycin (Sigma, Cat #P-7255), in
addition to 0.4 mg ml.sup.-1 Geneticin G418 and 0.1 mg ml.sup.-1
hygromycin. Several cell lines were identified that gave robust
levels of Ca.sub.v2.2 .alpha./.beta.3/.alpha.2.delta.1-mediated
current expression and one of these was selected for compound
profiling. Expression of all three subunits within this cell line
was continuously maintained by the inclusion of G418 (0.4 mg
ml.sup.-1), hygromycin (0.1 mg ml.sup.-1) and puromycin (0.62 .mu.g
ml.sup.-1). Cells were maintained at 37.degree. C. in a humidified
environment containing 5% CO.sub.2 in air. Cells were liberated
from the T175 culture flasks for passage and harvesting using TrpLE
(Invitrogen, Cat #12604-013).
[0429] Cell Preparation
[0430] Cells were grown to 30-60% confluence in T175 flasks and
maintained at 30.degree. C. for 24 hours prior to recording. Cells
were lifted by removing the growth media, washing with Ca.sup.2+
free PBS (Invitrogen, Cat #14190-094) and incubating with 3 ml of
warmed (37.degree. C.) TrpLE (Invitrogen, Cat #12604-013) for 6
minutes. Lifted cells were suspended in 10 ml of extracellular
buffer. Cell suspension was then placed into a 15 ml tube and
centrifuged for 2 minutes at 700 rpm. After centrifugation, the
supernatant was removed and the cell pellet was resuspended in 4.5
ml of extracellular solution.
[0431] Electrophysiology
[0432] Currents were recorded at room temperature (21-23.degree.
C.) using the IonWorks planar array electrophysiology technology
(Molecular Devices Corp.). Stimulation protocols and data
acquisition were carried out using a microcomputer (Dell Pentium
4). In order to determine planar electrode hole resistances (Rp), a
10 mV, 160 ms potential difference was applied across each hole.
These measurements were performed before cell addition. After cell
addition a seal test was performed prior to antibiotic
(amphotericin) circulation to achieve intracellular access. Leak
subtraction was conducted in all experiments by applying a 160 ms
hyperpolarizing (10 mV) prepulse 200 ms before the test pulses to
measure leak conductance. Test pulses stepping from the holding
potential (V.sub.H) of -90 mV to +10 mV were applied for 20 ms and
repeated 10 times at a frequency of 10 Hz. In all experiments, the
test pulse protocol was performed in the absence (pre-read) and
presence (post-read) of a compound. Pre- and post-reads were
separated by a compound addition followed by a 3-3.5 min
incubation.
[0433] Solutions and Drugs
[0434] The intracellular solution contained the following (in mM):
K-gluconate 120, KCl 20 mM, MgCl.sub.2 5, EGTA 5, HEPES 10,
adjusted to pH 7.3. Amphotericin was prepared as 30 mg/ml stock
solution and diluted to a final working concentration of 0.2 mg
ml.sup.-1 in intracellular buffer solution. The extracellular
solution contained the following (in mM): Na-gluconate 120, NaCl
20, MgCl.sub.2 1, HEPES 10, BaCl.sub.2 5, adjusted to pH 7.4.
[0435] Compounds were generally prepared in DMSO as 10 mM stock
solutions and subsequent 1:3 serial dilutions performed. Finally
the compounds were diluted 1:100 in external solution resulting in
a final DMSO concentration of 1%.
[0436] Data Analysis
[0437] The recordings were analyzed and filtered using seal
resistance (>40 M.OMEGA.), resistance reduction (>35%) and
peak current amplitude (>200 pA) in the absence of compound to
eliminate unsuitable cells from further analysis. Paired
comparisons between pre-compound and post-compound additions were
used to determine the inhibitory effect of each compound. The
concentrations of compounds required to inhibit current elicited by
the 1.sup.st depolarizing pulse by 50% (tonic pIC50) were
determined by fitting of the Hill equation to the concentration
response data. In addition the use-dependent inhibitory properties
of the compounds were determined by assessing the effect of
compounds on the 10.sup.th versus 1.sup.st depolarizing pulse. The
ratio of the 10.sup.th over 1.sup.st pulse was determined in the
absence and presence of drug and the % use-dependent inhibition
calculated. The data was fitted using the same equation as for the
tonic pIC.sub.50 and the concentration producing 30% inhibition
(use-dependent pUD.sub.30) determined.
[0438] The compounds of Examples 1 to 11, 13 to 24 were tested in
the hCa.sub.V2.2 assay and demonstrated the following pUD.sub.30
and pIC.sub.50 values. Compounds were tested in the form as
described in the Examples. All compounds tested have been tested
one or more times (up to 12 times). Variations in pUD.sub.30 and
pIC.sub.50 values may arise between tests.
[0439] The compounds 1 to 11, 13, 15 to 24 exhibited a pUD.sub.30
value of 4.5 or more than 4.5. The compounds 1 to 11, 13, 15 to 23
exhibited a pUD.sub.30 value of 5.0 or more than 5.0. The compounds
3, 4, 9, 11, 13, 16, 17 and 18 exhibited a pUD.sub.30 value of 5.5
or more than 5.5.
[0440] The compounds 1 to 11, 14 to 16, and 18 to 24 exhibited a
mean pIC.sub.50 value of 5.0 or less than 5.0. The compounds 1, 3
to 8, 10, 11, 14, 15, 18, 19, and 21 to 24 exhibited a mean
pIC.sub.50 value of 4.5 or less than 4.5.
* * * * *